- medication: 1

II Paper ID: 05d47dd5b46f86428de058db4ecc8bca76a9ad16 (Document no.: 219)

III Authors: Authors not available

Abstract not available

IV Sentences in paper containing search words:

INTRODUCTION The world is currently faced with a pandemic of novel coronavirus disease 2019 (COVID- 19) , which is caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) and has no vaccine or cure. It is predicted the development of a safe and effective vaccine to prevent COVID-19 will take one year to 18 months, by which time it is likely that several hundreds of thousands to millions of people may have been infected. Since coronaviruses causing COVID-19, Severe Acute Respiratory Syndrome (SARS), and Middle East Respiratory Syndrome (MERS) are able to suddenly transfer to humans from diverse animal hosts that act as viral reservoirs, there is a pressing need to develop methods to combat other potential coronaviruses that may emerge in the future [1] [2] [3] . A recent report further showed two strains (L and S) of SARS-CoV-2 with different genome sequences are circulating and likely evolving, further highlighting the need for a pan-coronavirus vaccination strategy 4 .The novel coronavirus causing COVID-19 belongs to a family of positive-sense RNA viruses, which typically infect the upper and lower respiratory tracks and cause disease by direct cytotoxic effects and the induction of host cytokines disease 5 . Therefore, we tested our approach using synthesized fragments of SARS-CoV-2, as well as with live H1N1 IAV. https://doi.org/10.1101/2020.03.13.991307 doi: bioRxiv preprint Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza Abbott et al. Through the use of crRNA pools targeting different regions of the same virus or different strains of coronavirus, this system could possibly buffer against viral evolution and escape, as well as enable rapid development and deployment against emerging viruses. When combined with an effective delivery platform, PAC-MAN is a promising strategy to combat not only coronaviruses including that causing COVID-19, but also a broad range of other genera of viruses. https://doi.org/10.1101/2020.03.13.991307 doi: bioRxiv preprint Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza Abbott et al. https://doi.org/10.1101/2020.03.13.991307 doi: bioRxiv preprint Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza Abbott et al. A total of ten groups (G1-G10) were tested for all 40 crRNAs (Supplemental Table 2 ). https://doi.org/10.1101/2020.03.13.991307 doi: bioRxiv preprint Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza Abbott et al. https://doi.org/10.1101/2020.03.13.991307 doi: bioRxiv preprint Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza Abbott et al. We argue that this approach, in which the lentivirus-derived RNA serves as a PAC-MAN target prior to lentivirus integration into DNA, mimics the crRNA targeting conditions that would apply for natural COVID-19 infection. We tested on a few selected crRNA pools, including the 3 best groups G4, G5, and G8 and one less efficient group G1, with a lentiviral multiplicity of infection (MOI) of 0.5. CRISPR PAC-MAN is able to inhibit IAV infection in human lung epithelial cells Since we do not yet have access to live SARS-CoV-2 virus, as a proof-of-concept we elected to test the CRISPR-Cas13d strategy on inhibiting H1N1 IAV, a RNA virus with a similar tropism as SARS-CoV-2 for respiratory tract epithelial cells. https://doi.org/10.1101/2020.03.13.991307 doi: bioRxiv preprint Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza Abbott et al. Manuscript 9 To test the efficiency of IAV-targeting crRNAs, we performed a screen of our crRNA pools in the stable Cas13d A549 cell line to determine which were most effective at inhibiting IAV infection (Fig. To verify Cas13d-mediated inhibitory effects on S4 and S6, we further tested at a lower MOI of 0.5, which showed better inhibition of IAV across samples (e.g., 79% reduction using crRNAs targeting S6; Fig. Cas13d PAC-MAN as a potential pan-coronavirus inhibition strategy In the past two decades, multiple variants of coronavirus, including those causing COVID-19, SARS and MERS, emerged from animal reservoirs and infected humans, each time causing significant disease and fatalities 1-3 . We found that just two crRNAs could target ~50% of coronavirus genomes, including those causing COVID-19, SARS, and MERS; 6 crRNAs were able to target ~91% of coronavirus genomes; and 22 crRNAs covered all sequenced coronaviruses with no mismatches (Fig. The ability to use a relatively small number of crRNAs to broadly target most or all coronavirus strains further highlights the uniqueness of our approach in contrast to traditional pharmaceutical or vaccination approaches.As a resource, we provide a map to show how our top 6 crRNAs (PAC-MAN-T6) targeted the coronavirus phylogenetic tree (Fig. Beta coronaviruses, the genus of viruses causing COVID-19, SARS, and MERS, were mostly covered by 3 crRNAs (crRNA 1-3) , while the other genera were covered by these three plus the remaining 3 crRNAs. https://doi.org/10.1101/2020.03.13.991307 doi: bioRxiv preprint Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza Abbott et al. https://doi.org/10.1101/2020.03.13.991307 doi: bioRxiv preprint Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza Abbott et al. While this strategy is a proof-of-concept and will require further testing using replication-competent SARS-CoV-2 viruses and validation in animal models before clinical tests in humans, it represents a unique approach to implement a rapid and broad antiviral defense in humans against emerging pathogens for which there are no effective vaccines.For both SARS-CoV-2 and IAV, we found highly conserved regions of the viral genomes to target with Cas13d. Remdesivir, created by Gilead Sciences (development code GS-5734), is an RdRP inhibitor that was developed to treat Ebola and Marburg virus infections and has been reported to have antiviral activity against MERS and SARS 36-38 . Remdesivir is already administered for COVID-19 through compassionate use requests, and clinical trials for its use against COVID-19 have already started in the U.S.(clinical trial ID NCT04280705) and China. Oseltamivir (also known as Tamiflu), is an antiviral medication for treating and preventing IAV that acts as an inhibitor of NA, thereby preventing the egress of new viral particles 39 . https://doi.org/10.1101/2020.03.13.991307 doi: bioRxiv preprint Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza Abbott et al. If the crRNAs targeting these viruses can be tested and validated before they ever infect humans, we can greatly accelerate the development of countermeasures for future emergent threats.In summary, our PAC-MAN strategy represents a potentially powerful new approach for inhibiting viral function and replication, and we envision it could be useful for a diverse array of circulating and emergent viral threats. the one with the fewest, shortest stretches of repeated nucleotides) was added to the set of minimal crRNAs. https://doi.org/10.1101/2020.03.13.991307 doi: bioRxiv preprint Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza Abbott et al. Twenty-four hours after transfection or 48 hours after transduction, cells were tested using flow cytometry on a Beckman-Coulter CytoFLEX S and qRT-PCR on a Biorad author/funder. An independent two-tailed T test was used to calculate the p values and statistics in Fig. https://doi.org/10.1101/2020.03.13.991307 doi: bioRxiv preprint Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza Abbott et al.

In [72]: keywords = ['host protease inhibitors;furin inhibitors, CoV papain-like protease, main CoV-2 protease inhibitors; broad anti-virals, plant anti-viral substances, ECE2 antagonists, channel blockers, lysosomal pH, autophagy modifiers, heat stress, ER stress, immune response busters, RNA polimerase inhibitors, interleukin-1 receptor, stop cytokine storm, transfusion of covalescent plasma, stem cells to recover damaged lungs']

In [73]: papers, rank_result = rank(keyords, must_have_words)

Traceback (most recent call last):

File "<ipython-input-73-05594a3fe21d>", line 1, in <module>

papers, rank_result = rank(keyords, must_have_words)

NameError: name 'keyords' is not defined

In [74]: must_have_words = None

In [75]: papers, rank_result = rank(keywords, must_have_words)

Traceback (most recent call last):

File "<ipython-input-75-9c0ef4091b35>", line 1, in <module>

papers, rank_result = rank(keywords, must_have_words)

File "<ipython-input-2-898fec40a25c>", line 201, in rank

results = search(term, must_have_word)

File "<ipython-input-2-898fec40a25c>", line 3, in search

searchsentence = searchsentence.lower()

AttributeError: 'list' object has no attribute 'lower'

In [76]: papers, rank_result = rank(keywords, must_have_words)

Traceback (most recent call last):

File "<ipython-input-76-9c0ef4091b35>", line 1, in <module>

papers, rank_result = rank(keywords, must_have_words)

File "<ipython-input-2-898fec40a25c>", line 201, in rank

results = search(term, must_have_word)

File "<ipython-input-2-898fec40a25c>", line 3, in search

searchsentence = searchsentence.lower()

AttributeError: 'list' object has no attribute 'lower'

In [77]: print_ranked_papers(rank_result, top_n=3, show_abstract=True, show_sentences=True)

RESULT 1: Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza

I Number of search words in paper:

- development: 15

- test: 11

- covid: 10

- vaccination: 2

- medication: 1

II Paper ID: 05d47dd5b46f86428de058db4ecc8bca76a9ad16 (Document no.: 219)

III Authors: Authors not available

Abstract not available

IV Sentences in paper containing search words:

INTRODUCTION The world is currently faced with a pandemic of novel coronavirus disease 2019 (COVID- 19) , which is caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) and has no vaccine or cure. It is predicted the development of a safe and effective vaccine to prevent COVID-19 will take one year to 18 months, by which time it is likely that several hundreds of thousands to millions of people may have been infected. Since coronaviruses causing COVID-19, Severe Acute Respiratory Syndrome (SARS), and Middle East Respiratory Syndrome (MERS) are able to suddenly transfer to humans from diverse animal hosts that act as viral reservoirs, there is a pressing need to develop methods to combat other potential coronaviruses that may emerge in the future [1] [2] [3] . A recent report further showed two strains (L and S) of SARS-CoV-2 with different genome sequences are circulating and likely evolving, further highlighting the need for a pan-coronavirus vaccination strategy 4 .The novel coronavirus causing COVID-19 belongs to a family of positive-sense RNA viruses, which typically infect the upper and lower respiratory tracks and cause disease by direct cytotoxic effects and the induction of host cytokines disease 5 . Therefore, we tested our approach using synthesized fragments of SARS-CoV-2, as well as with live H1N1 IAV. https://doi.org/10.1101/2020.03.13.991307 doi: bioRxiv preprint Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza Abbott et al. Through the use of crRNA pools targeting different regions of the same virus or different strains of coronavirus, this system could possibly buffer against viral evolution and escape, as well as enable rapid development and deployment against emerging viruses. When combined with an effective delivery platform, PAC-MAN is a promising strategy to combat not only coronaviruses including that causing COVID-19, but also a broad range of other genera of viruses. https://doi.org/10.1101/2020.03.13.991307 doi: bioRxiv preprint Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza Abbott et al. https://doi.org/10.1101/2020.03.13.991307 doi: bioRxiv preprint Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza Abbott et al. A total of ten groups (G1-G10) were tested for all 40 crRNAs (Supplemental Table 2 ). https://doi.org/10.1101/2020.03.13.991307 doi: bioRxiv preprint Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza Abbott et al. https://doi.org/10.1101/2020.03.13.991307 doi: bioRxiv preprint Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza Abbott et al. We argue that this approach, in which the lentivirus-derived RNA serves as a PAC-MAN target prior to lentivirus integration into DNA, mimics the crRNA targeting conditions that would apply for natural COVID-19 infection. We tested on a few selected crRNA pools, including the 3 best groups G4, G5, and G8 and one less efficient group G1, with a lentiviral multiplicity of infection (MOI) of 0.5. CRISPR PAC-MAN is able to inhibit IAV infection in human lung epithelial cells Since we do not yet have access to live SARS-CoV-2 virus, as a proof-of-concept we elected to test the CRISPR-Cas13d strategy on inhibiting H1N1 IAV, a RNA virus with a similar tropism as SARS-CoV-2 for respiratory tract epithelial cells. https://doi.org/10.1101/2020.03.13.991307 doi: bioRxiv preprint Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza Abbott et al. Manuscript 9 To test the efficiency of IAV-targeting crRNAs, we performed a screen of our crRNA pools in the stable Cas13d A549 cell line to determine which were most effective at inhibiting IAV infection (Fig. To verify Cas13d-mediated inhibitory effects on S4 and S6, we further tested at a lower MOI of 0.5, which showed better inhibition of IAV across samples (e.g., 79% reduction using crRNAs targeting S6; Fig. Cas13d PAC-MAN as a potential pan-coronavirus inhibition strategy In the past two decades, multiple variants of coronavirus, including those causing COVID-19, SARS and MERS, emerged from animal reservoirs and infected humans, each time causing significant disease and fatalities 1-3 . We found that just two crRNAs could target ~50% of coronavirus genomes, including those causing COVID-19, SARS, and MERS; 6 crRNAs were able to target ~91% of coronavirus genomes; and 22 crRNAs covered all sequenced coronaviruses with no mismatches (Fig. The ability to use a relatively small number of crRNAs to broadly target most or all coronavirus strains further highlights the uniqueness of our approach in contrast to traditional pharmaceutical or vaccination approaches.As a resource, we provide a map to show how our top 6 crRNAs (PAC-MAN-T6) targeted the coronavirus phylogenetic tree (Fig. Beta coronaviruses, the genus of viruses causing COVID-19, SARS, and MERS, were mostly covered by 3 crRNAs (crRNA 1-3) , while the other genera were covered by these three plus the remaining 3 crRNAs. https://doi.org/10.1101/2020.03.13.991307 doi: bioRxiv preprint Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza Abbott et al. https://doi.org/10.1101/2020.03.13.991307 doi: bioRxiv preprint Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza Abbott et al. While this strategy is a proof-of-concept and will require further testing using replication-competent SARS-CoV-2 viruses and validation in animal models before clinical tests in humans, it represents a unique approach to implement a rapid and broad antiviral defense in humans against emerging pathogens for which there are no effective vaccines.For both SARS-CoV-2 and IAV, we found highly conserved regions of the viral genomes to target with Cas13d. Remdesivir, created by Gilead Sciences (development code GS-5734), is an RdRP inhibitor that was developed to treat Ebola and Marburg virus infections and has been reported to have antiviral activity against MERS and SARS 36-38 . Remdesivir is already administered for COVID-19 through compassionate use requests, and clinical trials for its use against COVID-19 have already started in the U.S.(clinical trial ID NCT04280705) and China. Oseltamivir (also known as Tamiflu), is an antiviral medication for treating and preventing IAV that acts as an inhibitor of NA, thereby preventing the egress of new viral particles 39 . https://doi.org/10.1101/2020.03.13.991307 doi: bioRxiv preprint Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza Abbott et al. If the crRNAs targeting these viruses can be tested and validated before they ever infect humans, we can greatly accelerate the development of countermeasures for future emergent threats.In summary, our PAC-MAN strategy represents a potentially powerful new approach for inhibiting viral function and replication, and we envision it could be useful for a diverse array of circulating and emergent viral threats. the one with the fewest, shortest stretches of repeated nucleotides) was added to the set of minimal crRNAs. https://doi.org/10.1101/2020.03.13.991307 doi: bioRxiv preprint Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza Abbott et al. Twenty-four hours after transfection or 48 hours after transduction, cells were tested using flow cytometry on a Beckman-Coulter CytoFLEX S and qRT-PCR on a Biorad author/funder. An independent two-tailed T test was used to calculate the p values and statistics in Fig. https://doi.org/10.1101/2020.03.13.991307 doi: bioRxiv preprint Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza Abbott et al.

In [78]: print('Ranked papers (document numbers):', papers)

Ranked papers (document numbers): [219]

In [79]: keywords = 'host protease inhibitors;furin inhibitors, CoV papain-like protease, main CoV-2 protease inhibitors; broad anti-virals, plant anti-viral substances, ECE2 antagonists, channel blockers, lysosomal pH, autophagy modifiers, heat stress, ER stress, immune response busters, RNA polimerase inhibitors, interleukin-1 receptor, stop cytokine storm, transfusion of covalescent plasma, stem cells to recover damaged lungs'

In [80]: must_have_words = None

In [81]: papers, rank_result = rank(keywords, must_have_words)

Word not in dictionary: papain like

Word not in dictionary: anti virals

Word not in dictionary: ece

Word not in dictionary: anti viral

Word not in dictionary: cov

Word not in dictionary:

Word not in dictionary: interleukin

Word not in dictionary: covalescent

Found search words: ['furin', 'cytokine', 'antagonist', 'rna', 'cov', 'recover', 'stem', 'storm', 'lung', 'substance', 'heat', 'ph', 'receptor', 'host', 'blocker', 'broad', 'buster', 'plant', 'response', 'immune', 'lysosomal', 'stop', 'main', 'stress', 'polimerase', 'channel', 'protease', 'damaged', 'transfusion', 'er', 'plasma', 'autophagy', 'cell', 'inhibitor', 'modifier']

In [82]: print_ranked_papers(rank_result, top_n=3, show_abstract=True, show_sentences=True)

RESULT 1: OR-01. Distinct Regulatory Functions Are Defined by HLA-DR Expression on Human CD4 + CD25 high Treg Cells. OR-02. CNS Dendritic Cells Drive Naive T Cell Proliferation and Epitope Spreading in Relapsing Experimental Autoimmune Encephalomyelitis. OR-03. A New Spontaneous Mouse Model for Human DevicTs Disease. OR-05. Synovial Intracellular Citrullinated Proteins Colocalizing with Peptidyl Arginine Deiminase Are Pathophysiologically Relevant Antigenic Determinants of Rheumatoid Arthritis-Specific Humoral Autoimmunity

I Number of search words in paper:

- er: 7314

- cell: 3157

- ph: 1912

- immune: 850

- response: 718

- cytokine: 516

- stem: 364

- rna: 310

- receptor: 282

- main: 256

- plant: 153

- inhibitor: 126

- cov: 118

- plasma: 106

- lung: 81

- host: 76

- recover: 75

- stress: 29

- stop: 29

- broad: 23

- antagonist: 23

- heat: 18

- protease: 16

- blocker: 11

- channel: 11

- transfusion: 9

- substance: 7

- damaged: 6

- lysosomal: 3

- polimerase: 2

II Paper ID: dc2f210539245c7a03dcaa21c9305555d10ece43 (Document no.: 21371)

III Authors: C M Baecher-Allan, E Wolf, D A Hafler , S D Miller, E J Mcmahon, S L Bailey, H Waldner, E Bettelli, G Moorthy, D Baeten, R A Sobel, A Holz, H Wekerle, V K Kuchroo, C B Marta, N H Ruddle, A R Oliver, R Bansal, S E Pfeiffer, L De Rycke, A P Nicholas, T Cantaert, E Kruithof, J D Echols, B Vandekerckhove, E M Veys, F De Keyser

Abstract Although the CD4 + CD25 high regulatory T cell population represents only 2-3% of all peripheral blood CD4 T cells, it contains over one third of all class II expressing T cells in the peripheral blood. Highly purified, FACS-sorted DR + (~30% DR + CD25 high ) and DR-(~70% DR-CD25 high ) CD4 + CD62L high CD25 high Treg cells demonstrate equivalent suppressive activity in an anti-CD3 driven, in vitro dmicroT co-culture system. Both types of CD25 high Treg cells exhibit cell contact-dependent suppression, anergy, and expression of Foxp3 mRNA, at only slightly different levels.Substantial differences in the inhibitory nature of the DR + CD25 high and DR-CD25 high populations are uncovered when the cells are provided with different strength costimulatory signals. Upon CD2 costimulation, DR + CD25 high co-cultures exhibit a strong, early suppression of both proliferation and Th1/Th2 cytokine production, while DR-CD25 high co-cultures exhibit a much delayed suppression (late) that is accompanied by a preferential inhibition of Th1 cytokines (IFNg), and often an induction of IL-10 and IL-4. Importantly, IL-10 has contrasting effects on regulation by these two different types of Treg cells, underscoring another major difference between these two types of CD25 high Treg cells. Unlike the usual effect of IL-10 in reducing the immune response, IL-10 actually inhibits the suppression by DR + CD25 high and thus enhances co-culture responses. In contrast, IL-10 appears to be a component of the suppressive mechanism of the DR-CD25 high cells. Possibly due to this differential involvement of IL-10, the DR + CD25 high and DR-CD25 high populations cross regulate each other in vitro, and may do so in vivo as well. Importantly, these differences in the kinetics of suppression, Th1/ Th2 skewing, and involvement of IL-10 between the DR + CD25 high and DR-CD25 high populations are only seen when these two populations are studied as distinct populations. Thus it is apparent that the study of heterogeneous combined Treg populations would obscure possibly contrasting responses. It is possible that these different functional features may reflect a temporal order to the utilization of different regulatory subsets in vivo as the immune response switches from innate to adaptive immunity. Chronic progression of relapsing experimental autoimmune encephalomyelitis (R-EAE) in the SJL mouse is dependent on the activation of T cells to endogenous myelin epitopes, i.e. epitope spreading which plays a major pathologic role in disease progression. Using transfer of naive CFSE-labeled TCR transgenic T cells and mixed bone marrow chimeras, we show that activation of naive PLP139-151-specific T cells in SJL mice undergoing PLP178-191-induced R-EAE occurs directly in the CNS and not in the cervical lymph nodes, spleen or other peripheral lymphoid organs. Flow cytometric and histologic examination of the CNS during R-EAE revealed the infiltration of significant numbers of CD11c+ dendritic cells (DCs) (including myeloid, lymphoid and plamacytoid subsets) which are not seen in the healthy CNS. Functional examination of the antigen presentation capacity of APC populations purified from the CNS of mice with established PLP178-191-induced R-EAE shows that only F4/80-CD11c+CD45hi DCs efficiently present endogenous antigen resulting in the activation of naive PLP139-151-specific Tg T cells. In contrast, DCs as well as F4/80+CD45hi macrophages and F4/80+CD45lo microglia have the capacity to activate memory PLP139-151-specific Th1 cells. The current results indicate that naive T cells can gain access to the inflamed CNS, bypassing the need for activation in peripheral lymphoid sites, and that epitope spreading initiates principally within the CNS target organ. Further, activation of naive T cells is involved in chronic R-EAE is mediated by CNS DCs, not infiltrating macrophages or resident microglia. Consequently, blocking the recruitment or differentiation of DCs may be a viable target for inhibiting relapse and disease progression in murine MS models and possibly MS patients themselves. ; 5 These Authors Contributed Equally to the Work. Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by localized areas of inflammation and demyelination. MS can present itself as various clinical forms: a relapsing-remitting course, progressive course or unusual progression like in DevicTs disease in which lesions are found only in the optic nerve and in the spinal cord but not the brain. In addition, the first clinical presentation in a significant proportion of MS patient is often isolated optic neuritis (ON). The underlying immunological basis for different forms of MS and its association with other diseases like optic neuritis is not well defined.Experimental autoimmune encephalomyelitis (EAE) is a T cell mediated disease that shares many clinical and histological features with MS. However, to date, the development of ON has always been associated with EAE and there is no spontaneous animal model of DevicTs disease. Although myelin-specific Th1 cells are able to induce EAE in unimmunized mice, studies in both MS and EAE suggest that B cells and antibodies may play a role in demyelination. We have developed a TCR transgenic mouse, 2D2, specific for a minor protein of the CNS myelin called myelin oligodendrocyte glycoprotein (MOG) and have previously reported that a large proportion (47%) of these TCR transgenic mice developed spontaneous isolated ON. We have now crossed our TCR transgenic 2D2 mice to an IgH knock-in mutant mouse (Th) in which all the B cells are specific for MOG and produce MOG-specific antibodies. The knock-in mice do not spontaneously develop EAE. However, over 60 % of 2D2xTh mice, which express both MOG-specific T and B cells, developed a very early and severe form of EAE. Histological examination of the central nervous system of these animals reveal a selective distribution of the inflammatory foci and lesions restricted to spinal cord and optic nerve, a lesion pattern that is typical of DevicTs subform of MS. The importance of MOG specific T and B cells cooperation, and the role of antibodies in the development of DevicTs disease will be discussed.Antibody-induced demyelination is an important component of the pathology in multiple sclerosis (MS). In particular, antibodies to myelin oligodendrocyte glycoprotein (MOG) are elevated in MS patients and have been implicated as mediators of demyelination. The aim of our studies is to elucidate the mechanism of anti-MOG mediated demyelination. We show that antibody cross-linking of MOG in oligodendrocytes results in the repartitioning of MOG into lipid rafts, leading to changes in the phosphorylation of specific proteins, and culminating in rapid morphological alterations. Using proteomic analyses, we have identified 10 target proteins whose phosphorylation state is altered upon anti-MOG treatment. These proteins fell into functional categories related to the regulation of signal transduction, leading to cellular stress response and cytoskeletal instability. These changes were specific for anti-MOG; although cross-linking myelin associated glycoprotein (MAG) also instigates signaling modifications, these are distinct from those observed with MOG and did not result in oligodendrocyte morphological alterations. We next applied our findings to EAE models, analyzing antibodies to MOG that develop after immunization of C57BL/6 mice with MOG from rat or human origin. Interestingly, although these regimens result in EAE with similar anti-MOG antibody titers as evaluated by ELISA, only human MOG requires B cells for disease induction. Further, IgG to human, but not rat MOG, bound unfixed rodent oligodendrocytes and induced repartitioning of MOG into lipid rafts and morphological alterations. These data suggest a novel mechanism for antibody pathogenicity in B cell mediated EAE, and provide in vitro tools to determine whether an autoimmune antibody is pathogenic, and may be useful for evaluating the pathogenicity of antibodies in MS patients as an adjunct to diagnosis and treatment.

IV Sentences in paper containing search words:

Although the CD4 + CD25 high regulatory T cell population represents only 2-3% of all peripheral blood CD4 T cells, it contains over one third of all class II expressing T cells in the peripheral blood. Highly purified, FACS-sorted DR + (~30% DR + CD25 high ) and DR-(~70% DR-CD25 high ) CD4 + CD62L high CD25 high Treg cells demonstrate equivalent suppressive activity in an anti-CD3 driven, in vitro dmicroT co-culture system. Both types of CD25 high Treg cells exhibit cell contact-dependent suppression, anergy, and expression of Foxp3 mRNA, at only slightly different levels.Substantial differences in the inhibitory nature of the DR + CD25 high and DR-CD25 high populations are uncovered when the cells are provided with different strength costimulatory signals. Upon CD2 costimulation, DR + CD25 high co-cultures exhibit a strong, early suppression of both proliferation and Th1/Th2 cytokine production, while DR-CD25 high co-cultures exhibit a much delayed suppression (late) that is accompanied by a preferential inhibition of Th1 cytokines (IFNg), and often an induction of IL-10 and IL-4. Importantly, IL-10 has contrasting effects on regulation by these two different types of Treg cells, underscoring another major difference between these two types of CD25 high Treg cells. Unlike the usual effect of IL-10 in reducing the immune response, IL-10 actually inhibits the suppression by DR + CD25 high and thus enhances co-culture responses. In contrast, IL-10 appears to be a component of the suppressive mechanism of the DR-CD25 high cells. Possibly due to this differential involvement of IL-10, the DR + CD25 high and DR-CD25 high populations cross regulate each other in vitro, and may do so in vivo as well. Importantly, these differences in the kinetics of suppression, Th1/ Th2 skewing, and involvement of IL-10 between the DR + CD25 high and DR-CD25 high populations are only seen when these two populations are studied as distinct populations. Thus it is apparent that the study of heterogeneous combined Treg populations would obscure possibly contrasting responses. It is possible that these different functional features may reflect a temporal order to the utilization of different regulatory subsets in vivo as the immune response switches from innate to adaptive immunity. Chronic progression of relapsing experimental autoimmune encephalomyelitis (R-EAE) in the SJL mouse is dependent on the activation of T cells to endogenous myelin epitopes, i.e. Using transfer of naive CFSE-labeled TCR transgenic T cells and mixed bone marrow chimeras, we show that activation of naive PLP139-151-specific T cells in SJL mice undergoing PLP178-191-induced R-EAE occurs directly in the CNS and not in the cervical lymph nodes, spleen or other peripheral lymphoid organs. Flow cytometric and histologic examination of the CNS during R-EAE revealed the infiltration of significant numbers of CD11c+ dendritic cells (DCs) (including myeloid, lymphoid and plamacytoid subsets) which are not seen in the healthy CNS. Functional examination of the antigen presentation capacity of APC populations purified from the CNS of mice with established PLP178-191-induced R-EAE shows that only F4/80-CD11c+CD45hi DCs efficiently present endogenous antigen resulting in the activation of naive PLP139-151-specific Tg T cells. In contrast, DCs as well as F4/80+CD45hi macrophages and F4/80+CD45lo microglia have the capacity to activate memory PLP139-151-specific Th1 cells. The current results indicate that naive T cells can gain access to the inflamed CNS, bypassing the need for activation in peripheral lymphoid sites, and that epitope spreading initiates principally within the CNS target organ. Further, activation of naive T cells is involved in chronic R-EAE is mediated by CNS DCs, not infiltrating macrophages or resident microglia. Consequently, blocking the recruitment or differentiation of DCs may be a viable target for inhibiting relapse and disease progression in murine MS models and possibly MS patients themselves. Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by localized areas of inflammation and demyelination. MS can present itself as various clinical forms: a relapsing-remitting course, progressive course or unusual progression like in DevicTs disease in which lesions are found only in the optic nerve and in the spinal cord but not the brain. The underlying immunological basis for different forms of MS and its association with other diseases like optic neuritis is not well defined. Dendritic Cells Experimental autoimmune encephalomyelitis (EAE) is a T cell mediated disease that shares many clinical and histological features with MS. However, to date, the development of ON has always been associated with EAE and there is no spontaneous animal model of DevicTs disease. Although myelin-specific Th1 cells are able to induce EAE in unimmunized mice, studies in both MS and EAE suggest that B cells and antibodies may play a role in demyelination. We have now crossed our TCR transgenic 2D2 mice to an IgH knock-in mutant mouse (Th) in which all the B cells are specific for MOG and produce MOG-specific antibodies. However, over 60 % of 2D2xTh mice, which express both MOG-specific T and B cells, developed a very early and severe form of EAE. Histological examination of the central nervous system of these animals reveal a selective distribution of the inflammatory foci and lesions restricted to spinal cord and optic nerve, a lesion pattern that is typical of DevicTs subform of MS. The importance of MOG specific T and B cells cooperation, and the role of antibodies in the development of DevicTs disease will be discussed. Antibody-induced demyelination is an important component of the pathology in multiple sclerosis (MS) . We show that antibody cross-linking of MOG in oligodendrocytes results in the repartitioning of MOG into lipid rafts, leading to changes in the phosphorylation of specific proteins, and culminating in rapid morphological alterations. Using proteomic analyses, we have identified 10 target proteins whose phosphorylation state is altered upon anti-MOG treatment. These proteins fell into functional categories related to the regulation of signal transduction, leading to cellular stress response and cytoskeletal instability. These changes were specific for anti-MOG; although cross-linking myelin associated glycoprotein (MAG) also instigates signaling modifications, these are distinct from those observed with MOG and did not result in oligodendrocyte morphological alterations. We next applied our findings to EAE models, analyzing antibodies to MOG that develop after immunization of C57BL/6 mice with MOG from rat or human origin. Interestingly, although these regimens result in EAE with similar anti-MOG antibody titers as evaluated by ELISA, only human MOG requires B cells for disease induction. Further, IgG to human, but not rat MOG, bound unfixed rodent oligodendrocytes and induced repartitioning of MOG into lipid rafts and morphological alterations. These data suggest a novel mechanism for antibody pathogenicity in B cell mediated EAE, and provide in vitro tools to determine whether an autoimmune antibody is pathogenic, and may be useful for evaluating the pathogenicity of antibodies in MS patients as an adjunct to diagnosis and treatment. Synovial Intracellular Citrullinated Proteins Colocalizing with Peptidyl Arginine Deiminase Are Pathophysiologically Relevant Antigenic Determinants of Rheumatoid Arthritis-Specific Humoral Autoimmunity.ELISA in serum and synovial fluid and related to the anti-citrulline stainings in synovium and the presence of HLA-DR shared epitope.Results: Using different anti-citrulline antibodies, we confirm the RA-specific presence of synovial intracellular citrullinated proteins which are different from previously identified, non RAspecific deiminated proteins such as fibrin and vimentin. Additionally, the synovial intracellular citrullinated proteins detected in RA synovium determine directly the systemic ACPA levels as well as the local ACPA production in the joint. The relation between RA-specific intracellular citrullinated proteins and ACPA is dependent on the presence and load of the HLA-DR shared epitope.Conclusion: These data identify the RA-specific synovial intracellular citrullinated proteins as primary antigenic targets of ACPA in vivo and provide a pathophysiological rationale for the specificity of this autoimmune process in human RA.OR-06. K. A. Kuhn, 1 L. Kulik, 1 K. J. Braschler, 1 W. P. Arend, 1 V. M. Holers. 1 1 Immunology and Medicine, University of Colorado Health Sciences Center, Denver, CO, USA.Antibodies to citrulline-modified proteins have been shown to be specific and predictive markers of Rheumatoid Arthritis (RA) although the pathologic relevance of these antibodies has been unclear. Similar to that which has been observed in RA, in the murine collagen-induced arthritis (CIA) model of RA we have identified serum antibody reactivity specific for citrulline-modified proteins that precedes clinically apparent disease. To understand the role of antibodies to citrulline-modified proteins in disease, we examined whether tolerance to these antigens could protect mice from CIA. Mice were tolerized by intravenous administration of 0.3 mg of a citrulline-modified peptide, a noncitrullinated control peptide, bovine type II collagen (CII), or ovalbumin for 3 days. Three and 24 days after the final dose of tolerogen, mice were challenged with CII in complete FreundTs adjuvant. Mice tolerized with the citrulline-modified peptide demonstrated reduced disease severity compared to the control peptide and ovalbumin (2.4 F 0.8, 6.2 F 1.7, 6.4 F 1.3 , respectively, P b 0.05). These data suggest that immune responses to citrulline-modified proteins are important in the development of inflammatory arthritis. As a second demonstration of the pathogenic potential of antibodies to citrulline-modified proteins, we determined whether antibodies to these autoantigens could enhance disease. We then transferred D513 into mice alone and in combination with a submaximal dose of arthritis-inducing anti-CII monoclonal antibodies. In combination with anti-CII antibodies, D513 substantially enhanced disease severity (9.4 F 0.8 with D513 vs. 3.7 F 1.1 without, P b 0.001). Also, we created two additional monoclonal antibodies of the IgG class specific for citrullinated fibrinogen which also substantially enhanced disease severity in combination with anti-CII antibodies (8.3 F 1.2 and 8.3 F 0.7 , P b 0.05 compared to anti-CII antibodies alone). T to B Epitope Spreading Is Responsible for the Diversification of Autoantibody Responses within the Small Nuclear Ribonucleoprotein Complex. 1 1 Rheumatology and Immunology, University of Virginia, Charlottesville, VA, USA.Autoantibodies reactive against different polypeptides within the small nuclear ribonucleoprotein complex (snRNP) are often present in patients with systemic lupus erythematosus. Although the mechanisms for the initiation of anti-snRNP autoantibody responses are not known, it is well accepted that the complexity in this response is achieved through intra and intermolecular epitope spreading. We have established a model for intermolecular epitope spreading within the small nuclear ribonucleoprotein (snRNP) complex using the recombinant SmD protein.We have previously demonstrated that the initiating antigen and the interaction between the MHC and non MHC genes influences intermolecular epitope spreading. To elucidate the mechanisms for intermolecular epitope spreading within the snRNP complex, we immunized mice with synthetic peptides containing T cell epitopes on SmD. All peptides induced T cells reactive with the immunogens. In the A/J strain of mice peptides SmD 31-45 and SmD 52-66 consistently induced intermolecular epitope spreading to A-RNP. Consistent epitope spreading was not observed in mice immunized with peptide SmD [91] [92] [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] [104] [105] [106] [107] [108] [109] [110] . Although, all groups of mice had high titer of antibodies reactive with the peptide immunogens, antibodies capable of immunoprecipitating SmD antigen were minimally present. These data are in contrast to those obtained with whole protein immunization, wherein immunoprecipitating anti-SmD antibodies were readily generated. These data suggest that in SmD peptide immunized mice, a T to B cell epitope spreading was responsible for the generation of anti-A-RNP antibodies. Thus, if molecular mimicry was responsible for the initiation of anti-snRNP autoantibodies, our data indicate that a T cell mimic is sufficient to generate a diversified antibody response within the snRNP complex.We used pooled IgG immunoglobulins derived from 12 patients with primary SjogrenTs syndrome to screen a random peptide library to identify disease relevant autoantigen peptides. Among the identified peptides, one was recognised by 27/38 (72%) patientsT sera in an Elisa assay employing the solid phase peptide. This peptide was not recognized by sera of normal donors and of patients affected by other autoimmune diseases such as systemic lupus erythemathosus, rheumatoid arthritis and systemic sclerosis. Therefore the reactivity against this peptide appears to be confined within primary SjogrenTs syndrome patients.The identified peptide showed homology with the EBV encoded early antigen protein D. EBV infection has been associated with SjogrenTs syndrome. The same peptide shares similarity with the tear lipocalin, a protein highly expressed in tears and saliva, and with alpha-fodrin, a cytoskeleton protein considered an important autoantigen target in SjogrenTs syndrome.Anti-peptide antibodies affinity purified from patientsT sera recognize tear lipocalin in western blot. Moreover the same antibodies are able to bind alpha-fodrin, which is known to be cleaved in several fragments and to be exposed on the cell surface during apoptosis. Our findings suggest that tear lipocalin may be considered a novel and yet unidentified autoantigen in SjogrenTs syndrome. Recent studies suggest that increased T cell and autoantibody reactivity to lipids may be present in multiple sclerosis (MS) patients as compared to controls. We have created a 100-feature lipid ordered array containing duplicate spots of 50 brain, myelin, and microbial lipids and glycolipids that represent potential targets of the autoimmune response in MS. Using our lipid arrays, we tested cerebrospinal fluid from MS patients and other neurological disease (OND) controls for the presence of anti-lipid antibodies. Lipid array reactivity was quantified, and Significance Analysis of Microarrays (SAM) applied to identify lipids with statistically-significant differences in array reactivity between MS and control samples. Lipids with significant differences in array reactivity were ordered using a hierarchical cluster algorithm and displayed as heat maps using TreeView. Lipid arrays demonstrated increased autoantibody reactivity to lipids including sulfatides, 3b-hydroxy-5a-cholestan-15-one (an oxidized form of cholesterol), two separate forms of oxidized phosphatidyl-choline, lysophosphatidyl-ethanolamine, and sphingomyelin in MS patient CSF. Based on the array-determined anti-lipid antibody profiles, the patientsT samples clustered into groups of MS and OND controls. rosis (MS) are inflammatory demyelinating disorders of the CNS that share clinical and pathological characteristics. The role of autoantibodies as related to disease pathophysiology and diagnosis is unknown. To further characterize myelin autoantibodies in these diseases, we studied the humoral response to a panel of candidate autoantigens using protein arrays then confirmed these data using novel solid and solution phase antibody binding assays. Serum and CSF samples from patients with ADEM revealed robust binding to a wide range of autoantigens, but only a minor subset of samples from MS, encephalitis and normal controls demonstrated binding distinguishable from background. To confirm these data, two myelin antigens, myelin basis protein (MBP) and myelin oligodendrocyte glycoprotein (MOG), were further evaluated using traditional solid and novel fluid phase assays. Solid-phase binding to these antigens was observed in a minor subset of patients with MS and encephalitis, but solutionphase binding, a characteristic of higher affinity antibodies, was absent. Matched serum and CSF from patients with ADEM contained autoantibodies directed toward MBP and MOG. In contrast to their counterparts found in MS, these autoantibodies demonstrated robust solution phase binding. These data highlight a fundamental difference in the humoral response of these clinically similar diseases and reflects underlying differences in the pathogenesis of ADEM and MS.OR-11. Role of CD38 in Human B Cells: Evidence of a Functional and Physical Association with CD19.S. 1 1 Lab of Immunogenetics and CeRMS, Dept of Genetics, University of Torino, Torino, Italy.Human CD38 is a 45 kD surface glycoprotein endowed with ectoenzymatic and receptorial activities. The study of CD38 functions in T and NK cells indicated that the molecule bypasses its intrinsic structural inability to transduce signals through physical and functional associations with the TCR and CD16.Here we show that membrane localization is critical for CD38 signaling in human B cells. Membrane fractioning indicates that a relevant fraction of CD38 molecules (60-85%) is costitutively present in rafts in normal (tonsil) and neoplastic (Nalm-6, Raji, Daudi, Namalwa, Ramos, RPMI-8226 and U266) cells. Upon cross-linking all CD38 molecules translocate into the rafts, together with a fraction of CD19 but not of CD79a and b, suggesting that CD38 becomes physically associated with CD19.Lateral associations between the two receptors were confirmed in live cells using co-capping and biochemically with coimmunoprecipitation experiments.CD38-CD19 association also has a functional nature. Indeed, CD38 ligation transduces signals only in cells where CD19 is present and active. A formal proof of a functional interplay between CD38 and CD19 was obtained by the loss of CD38-mediated signals upon CD19 gene silencing.Lastly, all these events are independent of CD38 enzymatic activities which are present in all the cell lines analyzed and are unmodified by CD19 silencing.Together, these data further stress the notion that i) CD38 is a unique type of receptor working in synergy with the CD19 in B cells, and that ii) the enzyme and receptor functions of CD38 are distinct and independent. Toll-like receptors (TLRs) are evolutionarily conserved pattern-recognition receptors that are central to innate immunity. TLRs initiate innate responses to infection through selective recognition of conserved pathogen-associated motifs, which signal for cytokine induction. We have addressed whether endogenous signaling via TLRs directs the central nervous system (CNS) response to axonal injury. Stereotactic lesioning of axons in the entorhinal cortex causes axonal degeneration and rapid activation of CNS-resident cells (microglia and astrocytes) in specific regions of the hippocampus. We found that TLR2 was constitutively expressed in the hippocampus and was specifically upregulated by microglia in denervated zones prior to leukocyte recruitment. Detection of MyD88 and InBa mRNA suggested engagement of downstream signaling pathways. TLR2-deficiency reduced cytokine (TNFa) and chemokine (CCL3, CCL4, CXCL2 and CXCL10) levels, causing a delay in T cell infiltration. CCL2-driven macrophage infiltration was TLR2-independent, suggesting that the pathways which direct macrophage and T cell entry are differentially regulated by TLR2. Later expansion of the microglial population required TLR2 signaling, whereas astrocyte activation was unaffected by TLR2-deficiency. No responses were altered in TLR4-defective mice, arguing against endotoxin effects. Our findings suggest that TLR2 signaling directs the endogenous response to CNS axonal injury through selective regulation of early cytokine/chemokine expression that drives later cellular responses implicated in regeneration and repair. Innate Immune System Regulating This work was funded by the Multiple Sclerosis Society of Canada.OR-13. 1 There are frequent associations between microbial infections and autoimmune diseases in humans. An infection often exacerbates an ongoing autoimmune response leading to chronic inflammation, tissue destruction and degeneration of the corresponding target organ. The innate immune system is the first line of defense against pathogenic insult. Through Toll-like receptors (TLRs), the innate immune system has the ability to recognize pathogens or pathogenderived products and to initiate signaling cascades that trigger macrophages and dendritic cells to produce proinflammatory cytokines. This leads to the stimulation of the adaptive immune response. All known TLR ligands activate through the MyD-88 intracellular signaling pathway leading to the nuclear translocation of the rel-type transcription factor NF-kB and the activation of MAP kinases to induce gene expression of proinflammatory cytokines. We hypothesized that blockade of the innate immune system through TLRs would be a tangible method of attenuating the inflammatory cascade due to the inability of signaling through the MyD88dependent signaling pathway. In this study, we have investigated the involvement of TLR4 in the progression of experimental autoimmune encephalomyelitis (EAE), a prototypic model of multiple sclerosis. To bypass the need for TLR4 binding, we administered CpGs, the ligand for TLR9, to activate the MyD-88 signaling pathway. We show here that TLR4-deficient mice receiving a single dose of CpGs at the time of disease induction had an overall mean disease severity similar to wildtype mice. Therefore, manipulating the innate immune system through TLRs and the MyD-88 signaling pathway offers a unique opportunity to suppress destructive inflammatory responses and may provide a novel approach for the treatment of Th1-mediated autoimmune diseases. The transcription factor T-bet (T-box expressed in T cells) plays a major role in adaptive immunity. T-bet controls the development of both mouse and human Type 1 (Th1) T helper lymphocytes. However, the role of T-bet in the innate immune system has been largely unexplored. Here we demonstrate an essential function for Tbet in dendritic cells (DCs) in controlling inflammatory arthritis. We describe that collagen antibody-induced arthritis (CAIA) is a bipartite disease characterized by an early component, intact in Rag2 À/À mice, mediated through the innate immune system and a later phase influenced by the adaptive immune system. T À/À mice had markedly reduced joint inflammation at both early and late time points and Rag2 À/À /T-bet À/À double knockout mice were essen-Extra-cellular heat shock protein (HSP)-60 has been considered a pro-inflammatory bdanger signalQ. Yet, HSP60 can also down-regulate experimental immune arthritis and diabetes models by specific inhibition of Th1-like responses. We now report that HSP60 in vitro differentially modulates the expression of Th1/ Th2 transcription factors in human T cells: HSP60 downregulates T-bet, and NFATp, leading to decreased secretion of TNFa and IFNg and enhanced secretion of IL-10. These effects depended on TLR2-signaling, and could not be attributed to LPS or to other contaminants. In BALB/c mice, HSP60 in vivo inhibited the clinical, histological, and serological manifestations of ConA-induced hepatitis, associated with up-regulated T-cell expression of SOCS3 and GATA-3 and down-regulated T-bet expression. These results provide a molecular explanation for the effects of HSP60 treatment on Tcell inflammation via innate regulation of the inflammatory response. Toll Receptor Ligands Potently and Broadly Enhance the Immune Response of Immunodepressed Cutaneous T-Cell Lymphoma Patients. Patients with advanced cutaneous T-cell lymphoma (CTCL) exhibit profound defects in cell mediated immunity partially resulting from marked deficiencies in the numbers of peripheral blood dendritic cells (DCs) and in their capacity to make DC derived cytokines (IL-12, IL-15 and IFN-a). Because host immune function appears to play an integral role in mediating disease-controlling responses in CTCL, we investigated the effects of synthetic imidazoquinolines which have been recognized as immune stimulatory by virtue of activation of DCs following binding to Toll like receptor (TLR) 7 and 8. TLR 7, 8 and 7/8 binding compounds were cultured with freshly isolated peripheral blood mononuclear cells (PBMC) from patients with advanced CTCL (erythroderma with circulating malignant T-cells) and normal volunteers and a broad panel of immune functions assessed. The cytokine inducing effects were associated with marked activation of NK and CD8 T-cells assessed by CD69 expression and cytolytic activity. Furthermore, striking upregulation of CD80 expression on DCs and monocytes also occurred. Thus, imidazoquinolines exhibit the ability to potently and broadly enhance the immune response of patients with advanced CTCL. Such pre-clinical findings have typically been associated with significant clinical improvement when put into clinical practice and therefore have important implications for the potential enhancement of anti-tumor immunity among patients with advanced CTCL.OR-18. Beta-Adrenergic and Toll-Like Receptor 2 Signalling in Epidermal Keratinocytes Drive the Skin Immune Response to a Soluble Protein. dermatitis, contact hypersensitivity, lichen planus, alopecia areata and vitiligo. Here we show that preconditioning of the skin by hadrenergic antagonist and the Toll-like receptor 2 (TLR2) agonist S. Aureus peptidoglycan (PGN) results in increased local expression of the interleukin (IL)-1a (IL-1a) and IL-12 genes, which in turn instructs a T-helper 1 (Th1) adaptive response to a soluble protein antigen. On the contrary, when the TLR4 agonist E. Coli lipopolysaccharide (LPS) was used, the presence of the hadrenergic antagonist was not effective. These effects were consonant with the pattern of TLRs expression shown by epidermal keratinocytes but not by skin dendritic cells. As hadrenergic signalling defects together with S.Aureus infections are thought to serve as initiation and/or persistance factors for numerous Th 1-sustained inflammatory skin diseases, we might have disclosed at least part of the relevant pathogenetic mechanism.T Regs and Regulation of Immune M. I. Vargas-Rojas, 1 J. C. Crispin, 2 J. Alcocer-Varela. 3 1 Department of Immunology and Rheumatology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Mexico City, Mexico.During the last decade, the role of active suppression has gained an important place in the operational models of the immune response. Regulatory T (Treg) cells are now considered an essential part of the normal immune response and their absence or malfunction has been causally linked to disease in both animal models and clinical scenarios. Recently, our group and others, have reported that Treg cells are numerically deficient in patients with Systemic Lupus Erythematosus (SLE), a systemic autoimmune disease. Treg cells have been found to be functionally abnormal in other autoimmune diseases, however their functional competence has not been explored in patients with SLE. Thus, the aim of this work was to isolate Treg cells from patients with SLE in order to investigate if their suppressive capacity is normal. Fifteen patients with SLE (according to ACR criteria) and 15 age-and sex-matched controls were included. At the moment of the study, patients were not receiving corticosteroids nor immunosuppressive drugs; 4 patients had active disease. Treg cells (CD4 + CD25 ++ ) were quantified by flow cytometry. Results are expressed as the percentage of cells within total lymphocyte population. Intracellular IL-2 and IL-10 were quantified in CD4 + CD25 ++ and CD4 + CD25-cells. CD4 + CD25 ++ and CD4 + CD25-cells were isolated by magnetic separation. Suppressive capacity was quantified in 96 hour co-cultures: Treg cells were added to CD4 + CD25 + cell cultures stimulated with plate-bound anti-CD3 and soluble anti-CD28. Cell proliferation was calculated according to CFSE dilution. Proliferation results are expressed as an index that considers the number of cell divisions per cell (the cell proliferation method is the issue of another abstract). Treg cells were quantitatively lower in patients than in controls (active SLE 1.1 F 0.7; inactive SLE 0.71 F 0.8; controls 2.6 F 0.8, P b 0.01). As expected, intracellular IL-2 was exclusively observed in CD4 + CD25-cells. Conversely, intracellular IL-10 was regularly observed in CD4 + CD25 ++ cells. Treg cells obtained from healthy individuals did not proliferate neither in basal nor in stimulated conditions. Conversely, Treg cells from SLE patients proliferated when stimulated ( P b 0.05). When compared to cells obtained from normal controls, CD4 + CD25-cells from SLE patients proliferated more spontaneously, but less after stimulation ( P b 0.05). When cocultured, Treg cells from 11 out of 15 healthy controls inhibited z50% the proliferation of CD4 + CD25-cells; on the other hand, inhibition was only observed in cells from 3/15 patients with SLE ( P = 0.003). IL-2 Mediates Expansion but Not Maintenances of TGFB Induced Tregs in Inflammation: A Novel Role of IL-10.M. C. Fantini, 1 C. Becker, 1 I. Tubbe, 1 H. A. Lehar, 2 P. R. Galle, 1 M. F. Nerath. 1 1 Laboratory of Immunology, I.Medical Clinic, Johannes Gutenberg University, Mainz, Germany; 2 Institute of Pathology, Johannes Gutenberg University, Mainz, Germany.Regulatory T cells have been implicated in the maintenance of self tolerance in many animal models and human diseases. These cells have been subdivided in acquired and natural regulatory cells on the base of their origin. The first group includes regulatory cells generated in periphery as the results of antigen specific tolerization protocols, while the second is represented by naturally occurring CD4+CD25+ regulatory cells, generating in the thymus during the first days after birth. This last group of cells are characterized by the expression of the Winged Helix transcription factor FoxP3, which has been implicated in the direction of the genetic program determining the regulatory potential of naturally occurring CD4+CD25+ regulatory T cells. We have recently shown that FoxP3 and a regulatory phenotype can also be induced in CD4+CD25-naRve cells upon TGFh stimulation. In order to shed light on the in vivo physiology of Ti-Tregs we analyzed the expansion and phenotype stability of these cells in vivo. In brief, Ti-Tregs were adoptively transferred in SCID mice alone or together with colitogenic CD4+CD62L+ naRve cells in order to provide an inflammatory environment. Results obtained from these series of experiments indicate that Ti-Tregs depend for their survival and expansion on the presence of an ongoing inflammatory response as indicated by the loss of FoxP3 expression and regulatory capacity in Ti-Tregs transferred in SCID mice in absence of colitogenic cells. Further investigations of in vivo Ti-Treg requirements lead to the identification of exogenous IL-2 provided by the ongoing inflammatory response as the main responsible for Ti-Treg expansion and suppressive activity limited at the site of inflammation. Moreover, we have shown that IL-2 alone is not sufficient to maintain FoxP3 expression and the regulatory phenotype in Ti-Treg cells but that this requires the presence of IL-10.Therefore we propose TGFh induced regulatory T cells (Ti-Tregs) as a new class of acquired regulatory cells, highly inflammation dependent for their expansion and survival. This makes their action limited to the space where the immune response occurs and limited to the time this response lasts. The properties shown by Ti-Treg cells not only offer a new insight on how normally occurring immune responses can be physiologically controlled, but also offer the possibility to generate regulatory cells in vitro as a therapeutical tool circumventing the problem related to a prolonged and generalized state of immunodepression. Identification of Important Functional Domains of FOXP3 by Analysis of Mutations Present in Patients with IPEX Syndrome. T. R. Torgerson, 1 E. Gambineri, 2 S. Vijay, 1 S. Anover, 1 H. D. Ochs. 1 1 Pediatrics, University of Washington, Seattle, WA, USA; 2 Pediatrics, bA. MeyerQ ChildrenTs Hospital, Florence, Italy.FoxP3 is a member of the forkhead / winged-helix family of transcriptional regulators that has been shown to play a key role in the development and function of CD4 + CD25 + regulatory T cells in mice. In humans, defects in the FOXP3 gene lead to a disease of systemic autoimmunity that is characterized by severe autoimmune enteropathy, endocrinopathy, and skin disease known as IPEX (Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked). To define regions or domains of FOXP3 that may be functionally important, we have sequenced the FOXP3 gene in more than 60 patients with a clinical phenotype suggestive of IPEX syndrome. The remaining patients had normal FOXP3 sequences at the genomic DNA level but half of these had low FOXP3 mRNA expression. Clinically, patients with mutations in FOXP3 had more severe disease with a triad of enteropathy, dermatitis, and endocrinopathy often combined with other autoimmune phenomena whereas patients with normal FOXP3 sequences tended to have milder disease. There does not appear to be a geneotype/phenotype correlation between mutations in a particular region of the gene and a specific complex of symptoms. The mutations identified thus far, cluster in one of three regions: the carboxy-terminal forkhead domain, the leucine zipper, and the amino-terminal proline-rich domain, particularly near the 3V end of exon 1. Interestingly, no mutations have been identified in the zinc finger domain. We have introduced the mutations identified in patients into the FOXP3 cDNA and expressed them exogenously in cells to assess their effects on FOXP3 nuclear import, dimerization, DNA-binding and transcriptional control. Using this approach, we have identified regions in the carboxy-terminal portion of the protein that are required for nuclear import and DNA binding, regions in the central portion required for oligomerization, and regions important for the control of transcription.OR-22. GRAIL Expression Is Associated with the Biological Activity of CD25+ T Regulatory Cells. D. A. MacKenzie, 1 C. M. Seroogy. 1 1 Pediatrics, University of Wisconsin, Madison, WI, USA. CD25+ T regulatory cells (CD25+ Treg) are a subset of T cells with an anergic phenotype that suppress immune responses in an antigen-specific fashion by a poorly understood mechanism. To date the only specific gene marker for this subset of T cells is the transcription factor, Foxp3. We observe a link between CD25+ Treg cells and GRAIL, an E3 ubiquitin ligase necessary for the development of CD4+ T cell anergy in vivo. We hypothesize that GRAIL is important for the development and function of naturally occurring and induced CD25+ Treg cells. Several lines of evidence support this hypothesis: 1) GRAIL is expressed in naturally occurring CD25+ Treg cells at levels 10-fold greater than CD25-CD4 T cells, 2) a tolerizing immunization in vivo leads to the induction of longlived CD25 expressing antigen-specific tolerized T cells. Gene expression analysis of these cells reveals that GRAIL mRNA is upregulated (700-fold increase vs. CD25-tolerized cells; 100-fold increase vs. naturally occurring CD25+ Treg cells). Moreover, GRAIL expression is linked with Foxp3 expression strongly suggesting a suppressor phenotype in this induced tolerized population. As an initial step to understanding the role of GRAIL in CD25+ Treg cell suppressor function, we demonstrate that enforced expression of GRAIL in an antigen-specific T cell line is sufficient to convey a suppressor phenotype in vitro. These data link GRAIL expression to the biological activity of CD25+ Treg cells. Mechanistic studies are ongoing and will be discussed further.OR-23. Induction of CD4 + CD25 + Treg by Tolerance-Inducing Antigen-Presenting Cells Derived from Bone Marrow Cultures Initiated with Anti-CD200R2/3. Objective: The relatively ubiquitously expressed cell surface molecule CD200 delivers immunoregulatory signals following engagement of its receptor, CD200R. A family of CD200Rs has now been described by several groups, with the isoforms designated CD200R1-4. Little is known to date concerning the functional activity of CD200Rs expressed on cells in different tissues. We have used a number of isoformspecific anti-CD200R agonist mAbs to investigate the effect of signalling via CD200Rs expressed on splenic cells vs bone marrow dendritic cell (DC) precursors.Materials and Methods: We investigated the effect of agonist anti-CD200R mAbs in three independent functional assays: on the generation of alloimmunity in primary MLCs; on the generation of allostimulatory dendritic cells (DCs) from bone marrow cells cultured in vitro in the presence of (IL-4+GM-CSF); and on induction of Treg in ConA activated thymocytes in vitro.Primary MLC cultures were set up with C3H stimulator spleen cells, and mitomycin-c treated C57BL/6 stimulator cells. Cytokines were assayed in supernatants by ELISA at 40hrs, and CTL were assayed at day 5. In the second assay, bone marrow cells were cultured for 8 days with GMCSF and IL-4 with/without anti-CD200Rs, to generate DCs. DC maturation was induced overnight by LPS (1Ag/ml), and the mitomycin-c treated DCs were used to activate fresh splenocytes in MLC, or to induce Treg in splenocytes cultured for 48hrs with these DCs. T reg were also induced in ConA activated thymocytes cultured with anti-CD200R mAbs. Treg were assayed by FACS (for CD4 + CD25 + cells), using real-time PCR for FoxP3 expression, and by their ability to suppress CTL and cytokine production in a fresh primary MLC culture.Results: Anti-CD200R1 mAb (but not anti-CD200R2-4) suppressed cytokine production and generation of CTL directly in fresh MLC cultures. In contrast, addition of anti-CD200R1 caused no significant perturbation of development of allostimulatory DCs from bone marrow cultures with (IL-4+GMCSF). However, in the presence of anti-CD200R2/3 mAbs, no functional allostimulatory DCs developed from bone marrow cultures. Both populations of Treg were FoxP3 + and inhibited the antigen-specific OR-25. Regulatory T Cell Dysfunction and a Unique Autoreactive T Cell Population May Be Associated with the Development of Colitis in WASP-Deficient Mice.V. Cotta-de-Almeida, 1,2 F. Takeshima, 1,3 M. Maillard, 1, 4 P. Michetti, 4 S. B. Snapper. 1 1 Department of Medicine, Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA; 2 Department of Cell Biology, Oswaldo Cruz Foundation, Rio de Janeiro, RJ, Brazil; 3 Nagasaki University School of Medicine, Nagasaki, Japan; 4 Lausanne University Hospital, Lausanne, Switzerland.Background: Wiskott-Aldrich syndrome (WAS) is a severe immunodeficiency associated with autoimmunity, with up to 10% of patients developing inflammatory bowel disease (IBD). The majority of WASP-deficient (WKO) mice also manifest severe colitis. Objective:We aimed to determine whether IBD resulted from aberrant regulatory T cell function. Methods: Lymphoid organs obtained from WT and WKO mice were analyzed by flow cytometry and the number of CD4 + CD25 + regulatory T cells (Tregs) was determined. To directly assess the role of Tregs in colitis development, we adoptively transferred WT and WKO CD45RB hi (RB hi ) and CD45RB lo (RB lo ) CD4 + T cells alone or in combination into SCID recipients. Clinical scoring and histopathological analyses were performed. Results: WKO mice had reduced numbers of Tregs in the peripheral lymphoid organs. Interestingly, we also demonstrated decreased numbers of Tregs in the thymus. As expected, the adoptive transfer approach demonstrated that WT CD4 + RB hi cells induced colitis, which was blocked by the concomitant transfer of WT CD4 + RB lo cells. However, WT CD4 + RB hi cells transfered disease even in the presence of WKO CD4 + RB lo cells. We also found that WKO CD4 + RB hi cells are colitogenic, but they cause disease with late incidence and lower severity. Surprisingly, the co-transfer of WKO CD4 + RB hi and WKO CD4 + RB lo cells resulted in the development of more severe disease. Furthermore, WKO CD4 + RB lo cells alone, but not WT CD4 + RB lo cells, were able to induce colitis. Conclusions: Our data indicate aberrant development and function of Tregs in WKO mice. Furthermore, our transfer studies suggest the presence of unique colitogenic effector cells within the WKO CD4 + RB lo population. Aberrant Treg function and this unique autoreactive effector cell population may account for autoimmunity and IBD in WAS patients and WASP-deficient mice.OR-26. MHC Class II Controls CD4+, CD25+ Regulatory Tolerance to Allogeneic Transplants.G. Benichou, 1 S. K. Germana, 2 Y. Akiyama, 1 K. Tanaka, 1 C. LeGuern. 2 1 Surgery, MGH/Tranplant Unit, Boston, MA, USA; 2 Surgery, MGH/TBRC, Boston, MA, USA.MHC class II gene function has been closely associated with regulation of T cell immunity although the mechanism of their control remains unknown. We have previously demonstrated that the transfer of donor-type MHC class II genes in the bone marrow of miniature swine, recipients of subsequent renal allografts, induced immune tolerance which was not due to deletion of alloreactive T cells. In order to assess whether such tolerance mechanism would implicate the regulatory pathway, and notably CD4+, CD25+ regulatory T cells (T-regs), we examined the effect of somatic transgenesis of donor class II IAb genes in the bone marrow of CBA mice (H-2k), on the survival of subsequent C57BL/6 (H-2b) heart grafts. In this model, donor-specific tolerance as well as indefinite survival of fully allogeneic grafts were achieved, without the use of immunosuppression. Class IImediated tolerance spread to T cell responses to all graft antigens. Seventy seven percents of long-term accepted hearts, analyzed 160 days after transplantation, presented no signs of chronic allograft vasculopathy. In addition, a single injection of 160-day-tolerant Tregs in naive immunocompetent CBA mice, prolonged the survival of C57BL/6 grafts. These results indicate that class II-mediated tolerance affects the regulatory T-reg pathway likely through the emergence of class II-specific T-regs for suppression of the whole alloresponse, thereby achieving graft survival. They also provide a mechanism by which class II expression may control the regulation of T cell immunity to transplants. lipid metabolism, and inflammatory responses. Using Affymetrix oligonucleotide arrays we demonstrate that aP2 is also expressed in human bronchial epithelial cells (HBE), and shows a striking upregulation following stimulation of epithelial cells with the T helper 2 (TH2) cytokines IL-4 and IL-13. In HBE, aP2 was significantly down-regulated by the Th1 cytokine IFN-gamma. Upregulation by Th2-, and downregulation by Th1-cytokines strongly implicates aP2 as a participant in allergic inflammation. Consistent with this hypothesis, aP2 expression was markedly enhanced in bronchial epithelial cells from the lungs of mice undergoing allergic inflammation. Strong aP2 staining was also identified in the respiratory epithelium of human inferior turbinate biopsies. aP2 deficient mice were tested in the model of allergic airway inflammation and were found to be strongly protected, with reductions in airway eosinophilia, peribronchovascular inflammation and pulmonary Th2 cytokine production. Thus, aP2 is a critical regulator of allergic airway inflammation, and we are currently investigating its mechanism of action. Transcriptional Regulation of Autoimmune Diseases by Tumor Suppressor p53. Xiaochun Wan, Shi-Jun Zheng, Salah-Eddine Lamhamedi-Cherradi, Lingyun Xu, Brendan Hilliard, Youhai H. Chen. 1 Department of Pathology and Laboratory Medicine, School of Medicine University of Pennsylvania, Philadelphia, PA, USA.The tumor suppressor p53 regulates apoptosis, cell cycle, and oncogenesis. To explore the roles of p53 in autoimmune diseases, we studied autoimmune inflammation and innate immune function using C57BL/6 mice deficient in p53. We found that p53-deficient mice were more susceptible to experimental autoimmune encephalomyelitis (EAE) and streptozotocin-induced type I diabetes than control mice, and produced higher levels of IL-1, IL-6 and IL-12. Upon activation in vitro, p53-/-T cells and macrophages produced significantly elevated levels of inflammatory cytokines. The innate immune response of p53-/-macrophages to lipopolysaccharides and interferon-y was also significantly enhanced, which was accompanied with increased levels of total and phosphorylated signal transducer and activator of transcription (STAT)-1. These results indicate that p53 inhibits autoimmune inflammation and innate immunity through downregulating STAT-1 and pro-inflammatory cytokines.OR-29. The SjögrenTs Syndrome Associated Autoantigen Ro52 Is a RING-Dependent E3 Ligase That Suppresses Cellular Proliferation.A. Espinosa, 1 M. Hedlund, 1 S. Brauner, 1 V. K. Kuchroo, 2 M. Wahren-Herlenius. 1 Objective: To determine the function of Ro52. E3 ligases have been implicated in many cellular processes, including apoptosis, proliferation and signaling. Cbl-b, GRAIL and Itch, are regulators of important signaling pathways in immune cells. There is indirect evidence that there are other uncharacterized E3 ligases involved in regulating immune responses. Ro52 (Ro/SSA, Trim21, SSA1) is the main autoantigenic target in the autoimmune rheumatic disease SjfgrenTs syndrome. Methods and results: To investigate if Ro52 is an E3 ligase, ubiquitination assays were performed in vivo and in vitro. FLAG-Ro52, or FLAG-Ro52 lacking the RING domain (FLAG-Ro52DRING), was co-expressed with 6xHistidinetagged ubiquitin in HEK293 cells. Ubiquitinated proteins were purified with Ni-NTA resin and ubiquitinated Ro52 was visualized by immunoblotting with anti-FLAG antibody. FLAG-Ro52, but not FLAG-Ro52DRING, was polyubiquitinated suggesting that Ro52 is auto-ubiquitinated in a RING dependent manner. In vitro, Ro52 mediated polyubiquitination together with several ubiquitin conjugating enzymes, but most notably with UbcH6. Functional consequences of Ro52-mediated ubiquitination were investigated in a stably Ro52-transfected A20 B cell lymphoma cell line. Expression of Ro52-GFP in A20 B lymphoma cells resulted in a decreased proliferation in five independent clones compared to five GFP expressing A20 clones and the parental A20 cell line. Furthermore, when Ro52-GFP expressing A20 cells were activated with anti-CD40 antibody, there was an increased cell death in the Ro52 expressing A20 cells compared to the GFP expressing and parental cells. Conclusion: These data demonstrate that Ro52 is a novel RING-finger dependent E3 ligase, which inhibits proliferation and promotes activation induced cell death. Donor lymphocyte infusion (DLI) reliably induces durable remission in 75-80% of patients with relapsed CML following allogeneic bone marrow transplantation. To identify immunologic targets of the graft-versus-leukemia effect of DLI, we used CML post-DLI responder sera to screen a CML cDNA expression library. Two of the identified targets were derived from genes encoding proteins of 66 kD (CML66) and 28 kD (CML28). The development of high titer specific IgG responses against both antigens in DLI responders correlated with immune-induced remission. CML28 is identical to hRrp46p, a component of the human exosome, which is involved in the 3V processing of RNA. The human exosome contains known auto-antigens, such as PM/ Scl-100, an autoantibody target of patients with polymyositis or scleroderma. The present studies were undertaken to characterize the expression of CML28 and CML66 in primary normal and malignant hematopoietic tissues. Northern blots showed high-level expression in a variety of leukemia cell lines, but not in normal tissue, except for testis. Specific monoclonal antibodies to CML28 and CML66 were developed and utilized to detect antigen expression in leukemia cell lines and primary leukemia tissue on western blot and immunohistochemistry. Expression patterns were confirmed by antigenspecific real-time PCR. Both CML28 and CML66 were highly expressed in leukemic blasts from patients with AML and CML blast crisis, but barely detectable in normal bone marrow, normal peripheral blood, or leukemic cells from patients with stable phase CML. In contrast purified CD34+ progenitors from normal individuals and patients with stable phase CML expressed high levels of CML28 and CML66 transcript and protein. Immunohistochemical staining for CML66 confirmed rare staining of myeloid precursors in normal marrow and diffuse staining of myeloblastic cells in AML and blast crisis CML marrows. The expression patterns of CML28 and CML66 are similar to some other leukemia associated antigens, including the Wilms Tumor gene (WT1), and suggest that abundant expression in the malignant myeloid progenitor cell may be common to antigens that are targeted by the humoral immune response following DLI. The striking similarity between the expression patterns of CML28 and CML66 supports the notion that DLI exerts its curative effect by targeting antigens present in self-renewing malignant progenitor populations in CML. BAFF-but Not LT-Dependent B Cell Expansion Contributes to the Suppression of Lethal Intestinal Inflamation.Yasuyo Shimomura, 1 Emiko Mizoguchi, 2 Atul K. Bhan, 1 Atsushi Mizoguchi. 1 Expansion and accumulation of B cells are commonly observed in the inflamed intestine of IBD patients as well as experimental colitis models. Interestingly, the expanded B cells have been shown to play a regulatory role in certain intestinal inflammatory conditions (Immunity 2002,16:219; Gastroenterology 2004,126:115) . However, the molecular events leading to the inflammation-associated expansion of regulatory B cells have not been elucidated yet. Therefore, this study was designed to define the molecular mechanism using an acute colitis model in which colitis is induced by addition of 4% dextran sodium sulfate (DSS) in drinking water for 4 days. Interestingly, flow cytometric and immunohistochemical analyses showed a marked expansion of B cells in the colonic lamina propria (LP) during the recovery phase (day 8: four days after the cessation of DSS intake) but not in the acute phase (day 4) of DSS colitis. The expanded B cells represented a pre-mature phenotype with similarity to splenic transitional type 1 (T1) or T2 B cell subset. In addition, analysis of IgH A chain diversities showed that the B cells are polyclonally expanded. Surprisingly, lymphotoxin (LT) pathway, that is necessary for the development of GALT formation under normal conditions, was not required for the inflammation-induced B cell expansion as indicated by a marked expansion of B cells in the colon of DSS-treated LT a knockout (KO) mice. Therefore, to identify the factors involved in the B cell expansion, real-time PCR of purified B cells using a series of primer sets that can detect broad ranges of B cell-associated signaling molecules (237 molecules) was performed. Significant upregulations of BAFF-R (B cell-activating factor belonging to the TNF family-receptor), Btk (BrutonTs tyrosine kinase), Rac2 and CD40 were observed in colonic B cells at Day 8 compared to Day 0 and Day 4. The functional roles of these molecules in the inflammation-induced B cell expansion were then examined using the specific KO mice treated with DSS. During the recovery phase from DSS-induced acute colitis, expansion of B cells in the colon was not observed in BAFF-R deficient mice and reduced in Btk and CD40 KO mice. In contrast, absence of Rac2 did not affect the B cell expansion. BAFF-mediated activation of NF k B p52 has been shown to enhance the survival of B cells. Indeed, Western blot analysis showed an activation of NF n B p52 from precursor NF n B p100 in the Day 8 B cells. In addition, a marked increase in apoptosis of colonic B cells was observed in BAFF-R deficient mice compared to WT mice. Finally, to examine the functional role of B cells in DSS induced colitis, the colitis was induced in B celldeficient mice. Of note, over 60% of B cell-deficient mice died of the colitis until day 10, whereas over 90% of WT mice survived with a sign of recovery from the acute colitis. These studies indicate that intestinal inflammation-induced B cell expansion through BAFF but not LT pathway contributes to the suppression of acute lethal colitis.OR-32. Increased Constitutive Activation of NF-KB in Patients with Multiple Sclerosis.J. Yan, 1 B. Pat, 1 C. Winterford, 1 H. Inglis, 1 M. P. Pender, 1 J. M. Greer. 1 1 School of Medicine, The University of Queensland, Brisbane, QLD, Australia.Multiple sclerosis (MS) is characterized by inflammation and demyelination in the central nervous system (CNS). Current evidence suggests that MS results from autoimmune responses mediated by lymphocytes, which are activated in the peripheral lymphoid organs and migrate into the CNS. Recent studies suggest that the transcription factor NF-nB, might play an important role in the development of chronic autoimmune attack in diseases such as MS. NF-nB is normally found in the cytoplasm of the cell, but under inflammatory conditions it moves into the nucleus to initiate gene expression and produce more pro-inflammatory molecules. The activation of NF-nB is regulated by a group of inhibitor molecules, known as InB. Several of the InB family members have polymorphisms that have been associated with susceptibility to MS in Causasians. In immune cells from people with rheumatoid arthritis or asthma, NF-nB has been found with aberrant, constitutively nuclear localization and enhanced transcription of pro-inflammatory genes. It has been observed that NF-nB and InBa are co-localized in macrophage nuclei in active MS lesions. However, it is unknown if NF-nB is constitutively activated in the immune cells of MS patients. The aim of the current study was to investigate this question.Peripheral blood mononuclear cells (PBMC) were collected from 19 untreated patients with MS and 27 healthy controls. Some of the cells were fixed, pelleted, embedded in paraffin and sectioned for immunohistochemistry using antibodies specific for T cells, B cells or macrophages, and for the NF-nB p65 (RelA) subunit. The remainder of the cells were lysed, and cytoplasmic and nuclear fractions were purified from the lysate. These fractions were then analysed by polyacrylamide gel electrophoresis and immunoblotting using antibodies specific for the NF-nB p65 subunit and for InBa. The percentage of translocated NF-nB p65, defined as the relative amount of NF-nB p65 found in the nucleus divided by the total amount present in the whole cell, was determined for each sample.Sixty-eight percent of samples from untreated MS patients had increased NF-nB p65/RelA translocation to the nucleus, compared to 22% of PBMC from healthy controls (P b 0.002). Immunohistochemical studies confirmed the translocation of NF-nB p65 to the nucleus, and showed that, in most patient samples, the increased NF-nB activity was in T cells and monocytes rather than in B cells. In addition, the level of InBa protein was reduced in the cytoplasm of untreated MS patients compare to healthy controls, although not to a statistically significant degree.The results indicate that T cells and macrophages from untreated MS patients show higher levels of constitutively activated NF-nB than do healthy controls. Such activity may lead to enhanced transcription of pro-inflammatory genes and relate to the chronic nature of MS.investigated the contribution of the novel gene product, fibrinogen like protein 2 (fgl2) prothrombinase, in mediating immune injury in experimental and human acute allograft rejection.Method and Result: Using a mouse heterotopic cardiac transplant model, mouse fg12 (mfgl2)/fibroleukin mRNA transcripts and protein were highly expressed in macrophages, CD4 and CD8 positive T lymphocytes and endothelial cells in rejecting cardiac allografts in association with deposits of fibrin. While mfgl2 deficient mice rejected allografts at similar rates to littermate controls, survival of grafts from mfgl2 deficient mice were markedly prolonged and largely devoid of intravascular fibrin. Furthermore, treatment of wild type mice with a neutralizing anti-fgl2 polyclonal antibody ameliorated histological evidence for allorejection and intravascular fibrin deposition, and resulted in an increase in graft survival compared to graft survival from untreated mice or mice injected with an irrelevant antibody. To address further the relevance of human fgl2 (hfgl2)/fibroleukin in acute allograft rejection, we examined kidney biopsies from patients who had undergone renal transplantation. hfgl2 mRNA transcripts and protein were markedly expressed mainly in renal tubule cells, infiltrating lymphoid cells including macrophages, CD8+ + T cells, mature B cells (plasma cells) and endothelial cells. Dual staining showed fibrin deposition was localized mainly to blood vessels, in the glomerulus and interstitium and the lumen of tubules, and occurred in association with hfgl2 expression.Conclusion: These data collectively suggest that fgl2 accounts for the fibrin deposition seen in both experimental and human allograft rejection and provide a rationale for targeting fgl2 as adjunctive therapy to treat allograft rejection. 30123019 for Dr. XP Luo), from the Natural Science Foundation of China (NSFC), NSFC operating fund 30100171,and 30170846, and the Canadian Institutes for Health Research (CIHR) Grant #FRN33780. Glycolipid Mediated Activation of iNKT Cells Is Sufficient To Induce Airway Hyperreactivity Independent of Conventional CD4 T Cells. NK & NK T Cells dependence of AHR on iNKT cells producing IL-4 and IL-13. Eosinophils, B cells or IgE are not required for a-GalCer/PS30 induced AHR, since AHR develops in B cell deficient JHD mice and in mice treated with anti-IL-5 mAb to eliminate eosinophils. Moreover, MHC class II deficient mice, which lack conventional CD4 T cells but which have iNKT cells, show exaggerated glycolipid induced AHR, clearly demonstrating that conventional CD4 T cells are not required for AHR. Therefore, activation of pulmonary iNKT is necessary and sufficient to induce AHR in the complete absence of conventional CD4 T cells. We suggest that because iNKT cells are central and critical to the pathogenesis of AHR, therapies that target iNKT cells may be clinically effective in limiting the development of AHR and asthma.OR-36. The Involvement of CD1-Restricted NKT Cells in the Pathogenesis of Collagen-Induced and Antibody-Induced Arthritis.S. 1 1 Immunology, National Institute of Neuroscience, NCNP, Tokyo, Japan.Natural killer (NK) T cells are unique subset of T lymphocytes that express T cell receptor and a various NK receptors and produce a large amount of cytokines including IFN-g and IL-4 after stimulation with a glycolipid ligand such as a _ galactosylceramide (aGC). We have previously shown that administration of OCH, synthetic analogue of aGC, prevent collagen-induced arthritis (CIA) by preferentially inducing IL-4 production by NKT cells. However, the role of NKT cells in the natural course of arthritis models remains unclear. In the present study, we investigated the role of NKT cells in collagen-induced and antibody-induced arthritis. To induce collagen-induce arthritis, mice were immunized intradermally at the base of the tail with 100 Ag of bovine or chiken type II collagen (CII) emulsified with an equal volume of CFA. Mice were boosted by intradermal injection with the same antigen preparation on day 21. Starting from day 21, mice were injected intraperioneally twice per week with anti-CD1 antibody To induce antibody-induced arthritis, mice were injected either with the mixture of anti-CII monoclonal antibodies (mAbs) (Arthrogen-CIA mAb [Chondrex. Seattle, USA]) followed by LPS injection, or with serum from KRN T cell receptor transgenic mice crossed with non-obese diabetic mice (K/BxN). To detect NKT cells, cells were stained with a-galactosylceramide-loaded CD1 dimer and were analyzed using flowcytometry. Anti-CII antibodies were measured by enzyme-linked immunosorbent assay (ELISA). Histopathological examination was performed to evaluated arthritis. The number of NKT cells increased in the liver at the peak of the clinical course of CIA and antibody-induced arthritis. Next we induced in C57BL/6 (B6) mice and NKT cell deficient (Ja281 knockout) mice. The severity of arthritis was significantly reduced in NKT deficient mice compared to wild type B6 mice. The level of anti-collagen antibody was not different between these groups. The IgG1/IgG2a ratio of anti-CII mAb was elevated and IFN-g production from draining lymph node cells were reduced in NKT cell-deficient mice. To elucidate the role of NKT cells in the effector phase of arthritis, we next examined antibody-induced arthritis in NKT deficient mice and wild type B6 mice. The clinical arthritis score and pathological examination revealed that the severity of arthritis was significantly lower in NKT deficient (Ja281 knockout or CD1d knockout mice) compared to wild type B6 mice. CD1-restricted NKT cells play an important role in the pathogenesis of arthritis, particularly in the effector phase of arthritis. Considering the fact that Th2 skewing glycolipid ligand such as OCH inhibited the development of arthritis, NKT cells could be a new target for the treatment of rheumatoid arthritis.OR-37. Effects of Natural Killer (NK) Cells on Allogeneic Bone Marrow Transplantation.Swati Bhattacharyya, 1 Morton J. Cowan. 1 1 Department of Pediatrics, UCSF, San Francisco, CA, USA.Specific objectives of the study: In this stdy, we wanted to define the role of the NK cells in myeloablation in MHC mismatch condition. NK cells are capable of receptor mediated lysis of target cells that lack self class I MHC molecules. Thus it can be used effectively in an MHC I mismatched allogeneic bone-marrow transplant to create myeloablation instead of the T lymphocyte. The advantage of using NK cells is that it can achieve the creation of space without developing GVHD. Material, methods and results: Preliminary experiments were done to assess the myeloablative property of NK cells, where C57Bl/6 (B6) adult NK cells were injected i.p. This study showed that allogeneic NK cells destroys both erythroid and myeloid stem cells. In the next set of experiments we injected NK cells F bone-marrow from C57Bl/6 (B6) into 14 D old Balb/c fetuses. However due to toxicity we had to move to restrict the usage of NK cells postnatal only. Seven out of ten recipients of lin-BM and allogeneic NK cells had multi-lineage engraftment 8 weeks post transplantation. This indicate a) transplanted NK cells are playing important role in the engraftment and b) engraftment is occurring mostly through secretion of TGF-beta as the result of the addition of NK cells in transplanted cell mass. We used an immunodeficient T-B-NK+ scida -/-model to study the repopulation of the marrow following transplant with NK cells. We found significant repopulation of the T cells but significantly lower amount of B cells in blood. The genotyping PCR with thymocytes showed the successful reconstitution of thymus and T cells following NK cell + wt BM transplant in adult animals. The engrafted cells showed normal TCR rearrangement a feature lacking in the knockout animals. The bone-marrow analysis showed significant engraftment of B cells and normal IgG heavy chain rearrangement posttransplant unlike the scida where the development of B cells are halted at a very early stage. Conclusion: The NK cells can be used effectively as an cytotherapeutic myeloablative agent under immunosuppressed conditions. Exogenous IL-2 Promotes IL-5 Production by Human CD4 + NKT Cell Clones: The Role of IL-2 in the Immune Regulation.K. 1 1 Immunology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan.The ability of CD1d-restricted NKT cells to produce Th1 and Th2 cytokines has been well described. However, the potential of human NKT cells to produce cytokines under various immune responses needs yet to be delineated. We have analyzed the cytokine production of CD4 + NKT cell clones derived from human PBMC of healthy subjects and multiple sclerosis patients, and found that exogenous IL-2 would promote their IL-5 production. Methods: The NKT cell clones were generated by initially stimulating fresh PBMC with agalactosylceramide (aGC) or its analogue OCH. CD4 + NKT cells were then sorted based on the reactivity to anti-Va24, Vh11, CD4, and CD8 mAbs. To maintain the clones, Va24 + Vh11 + cells were sorted and then stimulated with PHA on monthly basis. The NKT cells were stimulated by aGC or OCH loaded on monocyte-derived immature dendritic cells, with or without exogenous IL-2. The supernatant was evaluated 48 hours later by cytomeric beads array (CBA). Results: Most clones produced IL-5, and the amount was higher than IL-4 in some cases. Exogenous IL-2 enhanced the cytokine production in response to the glycolipids in all the clones. Conclusion: Our data imply that, in the presence of IL-2, human CD4 + NKT cells could exhibit the potential to produce abundance of IL-5 in response to weak endogenous antigen stimulation, showing the interesting connection of IL-2 and the NKT cell-mediated immune regulation.OR-41. Specificity of NKT Cells Against GD3 Ganglioside.Dianna Y. Wu, Paul B. Chapman. 1 Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.We have shown previously that GD3, a ganglioside expressed on melanoma, can be presented by CD1 + antigen-presenting cells and that immunization with GD3 induces GD3-specific NKT cells. In the current study, we evaluated the specificity of the GD3-reactive NKT cells against other gangliosides that differ in the carbohydrate components that interact with the NKT cell T-cell receptor (TCR). By ELISPOT, we have found that a subset of NKT cells from GD3immunized mice also recognize GM3 but not GM2, GD2, or lactosylceramide. We interpret these results to mean that the presence of N-acetylgalactosamine (GalNc) inhibits interaction with the TCR since the NKT cells recognize GD3 (Glucose-galactose-[sialic acid] 2 ) but not GD2 (GalNc-glucose-galactose-[sialic acid] 2 ) or GM2 (GalNc-glucose-galactose-sialic acid). We also found that lactosylceramide (glucose-galactose) was not recognized by the GD3-reactive NKT cells suggesting that at least one sialic acid molecule is necessary for TCR binding. In conclusion, we have begun to characterize the epitope recognized by GD3-reactive NKT cells and find that the TCR of these NKT require glucose-galactose linked to at least one sialic acid for recognition, but that GalNc blocks TCR binding. CD4 + Invariant T Cell Receptor + NKT Cells and the Development of Bronchial Asthma in Humans. required for the development of airwayhyperreactivity (AHR), a cardinal feature of asthma. We now show that in human subjects with asthma, the majority of cells in bronchoalveolar lavage (BAL) fluid are not conventional CD4+ T cells, but rather CD4 + NKT cells expressing an invariant T cell receptor (iNKT cells). In the BAL fluid of the 11 asthmatic subjects, 55-85% of the CD3 + cells were iNKT cells, whereas in patients with sarcoidosis, another pulmonary inflammatory disease, b3% were iNKT cells. Similar results were obtained using immuno-fluorescence and confocal laser scanning microscopy of endobronchial biopsy specimens from patients with asthma and sarcodosis. Like iNKT cells in mouse models of asthma, the iNKT in the lungs of patients with asthma produced both IL-4 and IL-13, but very little IFN-g. In contrast, iNKT cells in the peripheral blood of all of our subjects (asthmatic, sarcoidosis and normal individuals) produced all three cytokines, suggesting the compartmentalization of Th2 subset of iNKT cells in the lungs of patients with bronchial asthma.Taken together, our studies demonstrate that the lungs of patients with asthma but not sarcoidosis contain predominantly CD4 + iNKT cells rather than conventional CD4 + T cells. These pulmonary CD4 + iNKT cells produce a cytokine profile known to amplify the inflammatory response in asthma strongly suggesting that iNKT cells play a central role in the pathogenesis of human asthma. In the mouse, distinct genetic variants of Tim1 were found to be associated with development of both Th2-biased immune responses and allergen-induced airway hyperreactivity. Here we examined the immunological function of TIM-1 using a specific monoclonal antibody (mAb), and demonstrated TIM-1 to be a potent T cell costimulatory molecule with a critical role in regulation of the immune system. TIM-1 is expressed on CD4 T cells early after activation, and expression is sustained preferentially in Th2 but not in Th1 cells. In vitro stimulation of CD4 T cells with a TIM-1 specific mAb in combination with T cell receptor activation markedly increased T cell proliferation, indicating that TIM-1 signaling provides a robust positive costimulatory signal to CD4 T cells. Administration of anti-TIM-1 mAb in vivo in combination with antigen strikingly enhanced production of cytokines, prevented the development of respiratory tolerance, and increased pulmonary inflammation. Our studies indicate that TIM-1 is a novel and significant costimulatory molecule, and altering TIM-1 function during the immune response could provide potent immunomodulatory therapies for a variety of immunological disorders. Genetics of Immune-mediated Diseases TLR, NK, Innate Immunity 10:30 AM-12:30 PM, 5/14/2005 OR-44 . In Vivo Homeostatic Proliferation of Naive CD4+ T-Cells Restrains the TCR Repertoire in Healthy Human Adults.S. Kohler, 1 U. Wagner, 2 M. Pierer, 2 S. Kimmig, 3 B. Oppmann, 1 B. Moewes, 1 K. Juelke, 1 C. Romagnani, 1 A. Thiel. 1 1 Clinical Immunology, DRFZ, Berlin, Berlin, Germany; 2 Medicine IV, University of Leipzig, Leipzig, Sachsen, Germany; 3 Molecular Biology, MPI for Infection Biology, Berlin, Berlin, Germany.Objective: Human CD31-central naive CD4+ T-cells have previously been shown to have post-thymically proliferated and to constitute the majority of naive CD4+ T-cells in the elderly. By taking advantage of this new phenotypic marker we wanted to analyse possible consequences of postthymic proliferation for the naive Th-cell pool and elucidate the driving force of this process in humans.Methods: The absolute numbers of peripheral blood CD31+ and CD31-naive CD4+ T-cells were determined in 25 donors of different age. Additionally, CD31+ and CD31-naive CD4+ T-cells of 9 healthy donors were highly purified and subjected to a detailed repertoire analysis by spectratyping. Finally RNA was isolated from purified CD31+ and CD31-naive CD4+ T-cells, cDNA prepared and subsequently Bfl/A1 expression analysed by RT-PCR.Results: We show here that absolute numbers of CD31-central naive CD4+ T-cells remain fairly stable in adult peripheral blood, excluding a thymus dependent regulation of the CD31-CD4+ central naive T-cell pool. On the contrary CD31+ thymic naive CD4+ T-cells decrease during ageing, implying a dependence on thymic function. Most importantly we demonstrate that CD31-central naive CD4+ T-cells isolated from healthy adults are characterised by a highly restricted oligoclonal T-cell receptor repertoire. In order to elucidate the driving force of this postthymic naive CD4+ T-cell expansion, we evaluated signatures of recent TCR engagement in purified CD31+ thymic naive and CD31-central naive CD4+ T-cells. Ex vivo RT-PCR analysis revealed upregulation of Bfl1/ A1 among CD31-central naive CD4+ T-cells, a gene which has been shown to be expressed exclusively upon TCR.Conclusion: Our results demonstrate for the first time that presumably TCR driven peripheral homeostatic proliferation of naive CD4+ T-cells in healthy human individuals causes a significant contraction of the peripheral TCR repertoire. Given the importance of a highly diverse repertoire for the ability to mount efficient immune responses, this age-dependent deterioration of CD4+ T-cell immunity could entail ageing-associated increased susceptibility to infection or cancer and decreased efficiency of vaccination. Moreover preferential expansion of self-reactive naive T cells could contribute to autoimmunity.Foxp3 is a transcription factor and its expression has been described as a unique marker for regulatory T cells (Treg). We have recently described that intragraft expression of latent TGFh is associated with stable kidney allograft function in monkeys no longer receiving immunosuppression. Thus we were interested if TGF-h expression and stable graft function correlated with intragraft Foxp3 expression. In addition, these tissues were stained for latent and active TGF-h. All animals with clinical rejection (serum creatinin of 200AMol or higher) and that showed significant graft infiltrates expressed Foxp3 in the nucleus in 5 to 20% of lymphocytic cells. There was one exception: one animal that rejected while still on CsA treatment did not show nuclear Foxp3 expression. In early biopsies (day 21-42 post transplantation) of animals treated with anti-CD40 and anti-CD40+CD86, only approximately 50% of the animals showed nuclear Foxp3 expression (5-10% of graft infiltrating cells). Most of these animals showed significant interstitial infiltrates without loss of graft function. Five animals were treated with ATG at the time of transplantation followed by anti-CD40+86 treatment. Only later when these animals also showed clinical graft rejection, Foxp3 could be found in the infiltrating cells. Animals (n = 6) that were on delayed CsA treatment (combined with anti-CD40+86) did not show expression of Foxp3, while Foxp3 expression was evident prior to the CsA treatment. Three animals that had stable graft function for more than 2 years after all immunosuppression was withdrawn showed low levels of nuclear Foxp3 expression or no nuclear Foxp3 expression at all. There was no direct correlation between the amount of either latent TGF-h or active TGF-h present in the grafts and the presence of nuclear Foxp3. We conclude that nuclear intragraft Foxp3 expression is not unique for tolerated grafts. Rather, Foxp3 positive cells (Treg) must be considered as part of the normal immune response during graft rejection. The nuclear expression of Foxp3 is inhibited by CsA and ATG treatment and this may indicate that these drugs prevent Treg development.if insulin is essential for development of autoimmune diabetes, we created NOD mice without native insulin 1 and 2 genes but with a mutated insulin B:9-23 sequence.The mutated insulin replaced tyrosine with alanine at B chain amino acid 16 (B16:Ala insulin). This mutation abrogates the autoreactivity of multuple insulin B:9-23-reacting T cell clones (e.g. BDC12.4.1) with preservation of hormonal activity. Four founder strains of B16:Ala insulin-transgenic mice were established directly in NOD mice and combined with insulin 1 and 2 knockout NOD mice.NOD mice lacking insulin 1 and 2 genes with the mutated insulin transgene did not develop anti-insulin autoantibodies, whereas 80% of mice bearing at least one copy of the native insulin 1 gene without the insulin 2 gene developed insulin autoantibodies (Pb0.0001). However sialitis was observed, suggesting that the protection from autoimmunity by deleting native insulin is organ-specific. Almost all mice with any native insulin gene sacrificed after 10 weeks of age had intra-islet insulitis with or without the B:16Ala transgene (n = 40/41, Pb0.01). All founder strains of B16:Ala-transgenic NOD mice developed diabetes and insulitis when a native insulin gene was also present, suggesting the preventive effect is dependent on the absence of the native insulin. Splenocytes from the protected transgenic native insulin null NOD mice showed delayed transfer of diabetes into NOD.SCID mice (50% transfer: 13.5 weeks), compared with standard NOD splenocytes transfer (50% transfer: 6.4 weeks, Pb0.02). Splenocytes from the diabetic NOD.SCID recipients showed immunologic memory and transferred diabetes into a second NOD.SCID mouse and this mouse developed diabetes at 5 weeks post splenocyte transfer.Our observation that native insulin null NOD mice with a mutation of insulin B:9-23 sequence abrogates anti-islet autoimmunity suggests that insulin is an essential autoantigen for type 1 diabetes of NOD mice, and insulin peptide B:9-23 is likely a critical determinant. Ability to transfer disease with splenocytes from protected mice is likely due to expression of native insulin B:9-23 sequence of the recipient and indicates that splenocytes from protected native insulin null mice are competent to rapidly transfer diabetes into host with appropriate target.yet undetermined factors. The New Zealand (NZ) mouse model, comprising the New Zealand Black (NZB) and New Zealand White (NZW) strains, spontaneously develops a systemic autoimmunity, and is considered to be an excellent model of SLE. Numerous SLE-linked loci have been identified throughout the NZ genome-the challenge now is to determine the genes that underlie these linkage regions and their role in SLE pathogenesis. To this end, we have assayed, utilising the Affymetrix MOE430 GeneChip system, global gene expression in both splenic CD19+ B-cells and CD4+ T-cells. Samples from lupusprone NZB and non-autoimmune BALB/c mice were investigated, along with two congenic mouse strains that carry NZBderived disease-linked regions on the BALB/c genome, namely distal chromosome 1 (Nba2) and proximal chromosome 4 (Nbwa2) .Differentially expressed genes between NZB and BALB/c were identified by combining the results of two methods-a fold-change analysis and a significance analysis of microarrays (SAM) . Genes that were observed as a result of both analyses were considered to be differentially expressed. Based on thresholds of F 2Â (compared to BALB/c) in the fold change analysis and 5% cutoff values for the SAM analysis, 559 genes and transcripts were differentially expressed between NZB and BALB/c B-cells and 758 genes and transcripts were differentially expressed between NZB and BALB/c T-cells.In an attempt to define disease susceptibility genes within the congenic intervals, the transcript profiles of genes that mapped within the congenic intervals were compared with those of BALB/ c. We successfully identified a number of differentially expressed genes specific to the congenic interval. For example, the interferoninducible (Ifi) gene cluster, sited on distal chromosome 1, showed a considerable degree of differential expression in both NZB and the Nba2 congenic strain compared to BALB/c. This replicates the findings seen in an investigation of gene expression in splenic tissue from NZB and C57BL/6.In relation to the Nbwa2 locus on proximal chromosome 4, upregulation of the BTB and CNC homology 2 (Bach2) gene was present in B-cells from the congenic strain when compared with BALB/c. Bach2 is a transcription factor that has key roles in B-cell class switching and the somatic hypermutation evident in the humoral immune response. We are defining sequence variants across the Bach2 gene in order to characterise its haplotype structure in inbred mouse strains.OR-48. Impact of the Lupus Susceptibility Locus, Sle1 on B Cell Tolerance.K. 1 1 Internal Medicine-Rheumatology, UT Southwestern Medical Center, Dallas, TX, USA.Purpose: Whereas B6 mice are autoantibody free; B6 mice rendered congenic for the NZM2410/NZW allele of the Sle1 lupus susceptibility interval develop high titres of anti-nucleosome antibodies. Hence Sle1 tips the balance from tolerance towards autoimmunity. These studies were designed to understand how Sle1 might breach B cell tolerance.Methods: B6.Sle1 mice were bred to HEL-Ig.mHEL or HEL-Ig.sHEL transgenic mice. These mice were then examined for breach in B cell tolerance using various methods including flow cytometry, serology, calcium flux analysis and in vitro cultures.Results: The presence of the HEL-Ig transgene bcured Q the Sle1 associated autoimmune phenotypes including splenomegaly (B6.Sle1.HEL-Ig 100 F 17 mg vs B6.Sle1 235 F 86 mg pb0.001, n = 8-22, aged 8-14 months), B (mfi I-A b B6.Sle1 866.6 F 142.4 units vs B6.Sle1.HEL-Ig 419. 1 F 80.38 units, n = 5-11, aged 3-7 months) and T cell activation (number of CD4 + CD69 + T cells: 9.187 F 3.736Â10 6 vs 4.222 F 1.264 Â10 6 n = 4-11), antinuclear antibody production (anti-DNA-histone IgG: B6.Sle1 144.1 F 46.21 U/ml vs B6.Sle1.HEL-Ig 18.62 F 2.820 U/ml, n = 3-11, aged 4-7 months, P = .0002) and glomerulonephritis.To study the impact on central deletion, Sle1 was bred to HEL-Ig.mHEL mice. B6.HEL-Ig.mHEL and B6.Sle1.HEL-Ig.mHEL (n = 6, each) mice had comparable diminution of splenic IgM a HEL +ve transgenic B cells and serum anti-HEL antibodies, indicating that Sle 1 did not abrogate central deletion of high avidity anti-self B cells.To study the impact of Sle1 on clonal anergy, Sle1 was bred to HEL-Ig.sHEL mice. In this model, although self-reactive B cells are allowed to escape to the periphery they are functionally anergised. Importantly, Sle1 breached tolerance in this model since B6.Sle1.HEL-Ig.sHEL mice had an increased number of B cells (18.66 F 3.791 Â10 6 vs 10.24 F 2.829 Â10 6 , p b 0.0006, n = 8-9, aged 3-6 months) and also had significantly higher levels of anti-HEL antibodies (39.70 F 5.643 U/ml vs 17.33 F 1.691 U/ml, n = 16-19 aged 3-7 months, P = 0.0013). Interestingly, some of the B6.Sle1.HEL-Ig.sHEL mice (3/10, aged 8-14 months) also produced anti-ssDNA IgM antibodies. Ex vivo overnight cultures of whole splenocytes demonstrated spontaneous activation of B6.Sle1.HEL-Ig.sHEL B cells in the absence of any stimulus and this activation was further enhanced in the presence of anti-IgM (n = 4, aged 3.5 months). Proliferation assays using CFDA-SE dilution revealed increased response of B6.Sle1.HEL-Ig.sHEL B cells to anti-IgM but not to sHEL(%undivided B cells: 28.33 B6.Sle1.HEL-Ig.sHEL vs 50.13% for B6.HEL-Ig.sHEL, n = 2 aged 3-6 months). To examine if presence of Sle1 rescues deletion of HEL-Ig B cells in sHEL mice, 25Â10 6 B6.HEL-Ig or B6.Sle1.HEL-Ig B cells were adoptively transferred into sHEL mice. However there was comparative deletion of either B cells.Conclusions: These findings support the conclusion that though Sle1 may not have the capacity to thwart central deletion of high avidity anti-self B cells, it certainly abrogates effective danergizationT of low-avidity anti-self B cells. The molecular bases for these differences are currently being examined.The aim of this study was to determine whether INS-VNTR polymorphisms modulate functional phenotype of the T cell response to P-Ins in subjects with high risk DR4-haplotype, with (T1D patients and Ab+ subjects) or without (control subjects) betacell autoimmunity. All subjects were typed for INS-VNTR class I and class III alleles. Peripheral blood lymphocytes and CD4+ T cell subsets (CD45RA+, naiive and recently primed and CD45RA-, memory) were stimulated with immunodominant P-Ins73-90 epitope, and cytokine secretion (Th1:IFNg, TNFa, IL-2, and Th2:IL-4, IL-5, IL-10) was determined. Our analysis reveal the predominance of CD4+CD45RA+IL-10hi cells in subjects with protective VNTR class III alleles, but not in subjects with VNTR class I alleles. CD4+CD45RA+IL-10hi T cell phenotype has been associated with regulatory function in subjects with T1D, and in experimental models of autoimmunity.Our analysis show, for the first time that transcriptional effects of VNTR genes in subjects with high risk DR4-haplotype affect the selection of P-Ins specific T lymphocytes in the periphery and influence predisposition to T1D. A Toll Receptor Pathway Polymorphism Affects Susceptibility to Inflammatory Bowel Disease. Inflammatory bowel disease (IBD) is a complex genetic disorders caused by a combination of genetic and environmental factors. Recent evidence has implicated a component of the innate immune system in the pathogenesis of IBD; mutations in NOD2/ CARD15 in humans have been associated with susceptibility to IBD. These data prompted us to undertake a thorough investigation of one innate immunity pathway involved in extracellular pathogen-associated molecular pattern recognition: the Toll-like receptor pathway. We selected 18 genes (the TLR gene family as well as components of downstream signalling pathways) and performed a haplotype-based association analysis for each gene. This assessment of the common genetic variations found in the TLR pathway reveals that there is suggestive evidence for association of polymorphisms in IRAK2, TIRAP, TLR3, and TLR4 to IBD in a screening Canadian population of 161 IBD trios and 114 cases and 68 controls. However, these results were not replicated in an independent Belgian sample collection of 104 IBD trios and 610 cases and 383 controls. A second replication effort consisting of 1000 IBD trios and 400 IBD tetrads will be concluded shortly and should allow us to make definitive conclusions regarding the observed associations. Two polymorphisms previously associated with IBD (Asp299Gly in TLR4 and an NF-kB promoter indel) were also assessed using meta-analyses of the published studies and our study populations. The NF-kB polymorphism failed to show definitive association in our metaanalysis; however, we definitively show that the 299Gly allele of TLR4 is associated with susceptibility to IBD and explore the phenotypic associations of this allele.OR-51. Warnatz, 1 U. Salzer, 1 S. Gutenberger, 1 M. Schlesier, 1 B. Grimbacher, 1 H. H. Peter, 1 H. Eibel. of Rheumatology and Clinical Immunology, University Clinic, Freiburg, Germany.Introduction: BAFF-R is a member of the TNF-R superfamily and is expressed mainly on mature B cells. Both BAFF-/-(synonym:Blys) and BAFF-R-/-mice exhibit a severe defect in peripheral B cell homeostasis indicating a prominent role of BAFF-R/BAFF interaction in the peripheral B cell development of the mouse. Hypogammaglobulinemia, disturbed germinal center formation and impaired antibody responses in BAFF-deficient mice rendered BAFF and its receptor clear candidate genes in the search for the etiology of CVID. Here we report the first patient with a homozygous mutation of the BAFF-R causing the clinical phenotype of CVID.Methods: 50 Patients were screened for surface expression of BAFF-R by flowcytometry. A suspected defect was confirmed by genomic DNA sequence, RT-PCR and Western blot analysis. Extensive immunologic phenotyping of peripheral blood cells was performed. Patient-derived B cells were compared with normal controls by in vitro activation via BCR F BAFF.Results: A 60 yr old patient with hypogammaglobulinemia (IgG 0,6g/l, IgA 5,3 g/l, IgM 0,6 g/l) and no family history of immunodeficiency was identified by FACS analysis to express no BAFF-R on B cells. The immune phenotype was distinct and may permit to screen for BAFF/BAFF-R deficiency. Besides recurrent pneumonias he suffered from an unusual fungal infection of his upper respiratory tract.Conclusion: The first patient with a genomic BAFF-R defect confirms the role of BAFF as a master regulator of peripheral B cell homeostasis also in humans. The immune phenotype of this patient may allow the identification of patients with related defects.Supported by DFG grants: a) SFB620 TPC1; b) SFB620 TPC2; c) SFB620 TPA2. Purpose: CTLA4 (Cytotoxic T Lymphocyte Associated Protein 4) may have widespread significance in the aetiology of several autoimmune diseases (AID). It is a good candidate gene for SLE because it is part of a suggestive linkage interval to lupus at 2q33, in two genome-wide linkage scans. Linkage to this region, and associations with different polymorphisms in CTLA4, have also been reported for type I diabetes (T1D), GravesT disease and coeliac disease. Functionally, CTLA4 is important in the inhibition of CD28-mediated T cell activation, through cooperation with two T cell co-stimulatory molecules, CD28 and ICOS. These key regulators of T cell activation are encoded in a 300kb region homologous to the Bxs1 lupus linkage interval on mouse chromosome 1 in BXSB mice. There are reported associations in several AID with polymorphisms in the 3V UTR, exon 1 and 3V flanking region of the gene. However, these results in both SLE and other AID, are inconsistent, largely being small frequencies between Africans and Europeans to warrant potential inclusion in our map. Experimental validation involved genotyping new population samples by the 5V nuclease assay (TaqMan Assayson-Demand) or by primer-oligo base extention assay resolved by MALDI_TOF mass spectrometry on a chip (MassARRAY Sequenom) . Markers were chosen if they were genotyped successfully in at least 20 West Africans and 20 European Americans, were in Hardy-Weinberg equilibrium in the parental populations, had a minimal level of ancestry informativeness (Shannon Information Content (SIC)N0.035 out of a maximum of 0.709) and were similar in frequency within continents. We are currently using a subset of the markers in a validated map (1, 536) to screen for risk factors for disease in 742 patients with multiple sclerosis and 996 with prostate cancer. The hallmark of atopic allergy is production of allergenspecific IgE, which in turn requires the cytokines interleukin (IL)-4 and IL-13, products of allergen-specific Th2 type cells. In addition to being responsible for activation and release of inflammatory mediators by mast cells and basophils, allergenspecific IgE is able to form complexes with the allergen, which can be efficiently processed and presented by IgE-receptor expressing antigen presenting cells (APC). Capture and presentation of the allergen via this mechanism has been shown to result in 100-fold reduction in the concentration of allergen required to trigger T-cell activation. Binding of allergen-IgE complexes to the surface of human B lymphocytes has been demonstrated previously by flow cytometry using fluorescently labeled anti-IgE antibodies. We have now investigated the binding of allergen-IgE and allergen-IgG1 complexes to the surface of splenic B-cells isolated from BALB/c mice. Polymorphisms in Methods: CD19 + B-cells were isolated from the spleen of naive BALB/c mice using magnetic beads (Miltenyi) . Serial dilutions of sera from ovalbumin-sensitized mice (immunized by intraperitoneal injection), or from naive (untreated) controls, were incubated alone or in the presence of 3 or 5 Ag of ovalbumin (OVA) for 1 hour at 37 8C. Subsequently, B-cells were added at 5 Â 10 5 / sample and incubated for 1 hour at 4 8C. Following incubation with sera/allergen, B-cells were washed twice with PBS containing 1% bovine serum albumin (BSA) and stained on ice for 45 minutes with monoclonal fluorescently-labeled anti-IgE and anti-IgG1 antibodies. In additional experiments fluorescently-labeled OVA was used to reveal binding of OVA/antibody complexes. Cells were analyzed after washing using a FACScalibur flow cytometer. For each sample a minimum of 5000 cells was analyzed.Results: In the absence of serum or allergen, a small percentage of B-cells was IgE and IgG1 positive. Incubation of B-cells with allergen (OVA) and sera from OVA sensitized mice at higher serum concentrations (40-80%) resulted in a marked increase in IgE + B-cells (up to 40% increase) and IgG1 + B-cells, which was already detectable at low serum concentrations (b1%). Binding of both IgG1 and IgE was dependent upon the presence of both allergen and allergen-specific antibodies since incubation with sera from OVA-sensitized mice and an irrelevant allergen, or from naive mice and OVA did not result in a similar increase in antibody positive B-cells. Furthermore, the binding of OVA-IgE, but not OVA-IgG1, complexes was shown to be inhibited markedly by treatment with anti-CD23 (FceRII) antibody in vitro.Conclusion: These data demonstrate that mouse splenic B-cells bind allergen-specific IgE through the low affinity IgE receptor (FceRII), with efficient binding only occurring in the presence of allergen-IgE complexes. This may form the basis of an approach for the characterization and identification of IgE containing sera.OR-55. Novel Candidate Markers for Multiple Sclerosis Using Phage cDNA Display.V. Somers, C. Govarts, K. Somers, P. Stinissen. 1 Biomedisch Onderzoeksinstituut (BIOMED), Limburgs Universitair Centrum (LUC)/Transnational University Limburg (tUL), Diepenbeek, Belgium.Multiple sclerosis (MS) is a chronic, inflammatory disease of the central nervous system, characterized by the presence of focal lesions resulting from myelin breakdown. In the past few years, an important contribution of B cells and autoreactive antibodies has been demonstrated in the pathogenesis of MS. To fully explore the complex information present within the antibody repertoire of patients, we have developed a novel and powerful molecular approach dSerological antigen selectionT, which involves the display of a cDNA expression library on filamentous phage and subsequent selection on patient IgG. The aim of this study was to apply the SAS technology to identify antigens that are specifically recognized by antibodies (IgG) present in the cerebrospinal fluid (CSF) of MS patients. First, we constructed a cDNA display library by cloning a normalized cDNA library prepared from active chronic MS plaques, with varying degrees of demyelination and inflammatory activity (Soares et al, 1994) for expression as a fusion protein with a filamentous phage minor coat protein, pVI. Parallel selections were then performed on 2 pools of CSF (n = 10) from relapsing-remitting MS patients. Affinity selections revealed a panel of 9 different clones reactive with the first CSF pool. A detailed serological analysis of the 9 different antigens on a large panel (n = 100) of individual patient and control CSF showed exclusive reactivity to MS patient CSF for seven different antigens. Sequence analysis revealed that these clones have never been associated with MS. Antigenic cDNAs from the second pool of CSF are currently under investigation. In conclusion, our findings demonstrate that this novel molecular approach is useful to identify novel candidate antigens in MS that can be used as diagnostic markers, and can be used to study the humoral immune response in MS. OR-56. Phenotypic and Functionnal Ex Vivo Profiling of Human CMV Specific CD8+ T-Cell Responses after Allo-HSCT.V. Bajzik, 1 N. Dulphy, 1 S. Saada-Duchenoy, 1 L. Leca, 1 C. Scieux, 3 E. Gluckman, 2 G. Socie, 2 D. Charron, 1 A. Toubert, 1 H. Moins-Teisserenc. 1 Recognition of viral antigen by the immune system induces a coordinate number of changes in lymphocyte subsets. This involves changes in the expression of cell surface molecules, in lymphocyte migratory properties and in the ability to proliferate and exert T-cell mediated cytotoxicity. Primary infection with human Cytomegalovirus (hCMV) is followed by lifelong persistence with viral latency in cells of the myeloid lineage. CMV specific CD8-T cells are maintained at a very high frequency in healthy CMV carriers, reflecting a permanent control of CMV reactivation at a subclinical level by the hosts immune response. Because primary CMV infection is usually clinically silent, little is known about the longitudinal evolution of specific T cells during the course of antigenic challenge. Due to the latency of the immune reconstitution, patients with allogeneic haematopoietic stem cell transplantation (allo-HSCT) are at high risk of CMV reactivation during the first three months. We have designed a strategy to follow the CMV reactivation in this context, combining weekly determination of the viral load with the quantification of CMV specific immune responses. We took advantage of such monitoring to explore the complex host-pathogen relation during the course of infection and latency. We focused on the immune dominant response against the tegument protein pp65 and characterized the CD8+ T cell response using a combination of phenotypic (HLAclass I tetramers) and functional (ELISPOT) assays. Twelve patients were selected for their ability to mount a CMV response of more than 2% of total CD8+ T cells. Specific T cells were detectable at the time of reactivation as early as 34 days after allo-HSCT and underwent a phase of expansion with stabilization of the response depending on the intensity of antigenic challenge. In recipient from a seronegative donor, CD27+CD28+ and CD27+CD28-early/intermediate phenotypes predominated during the first wave of reactivation. This was rapidly followed by a progression though to CD27-CD28-late phenotype. Although the CD45RA molecule was observed on most late phenotypes, CD45RA and CD45RO expression did not correlate with the 3 stages defined by CD27 and CD28 expression. In patients who reactivated twice or more, an enrichment of CD8+T cells with different phenotypes was observed, consistent with different stages of differentiation. All CD8+T cells were perforine positive whereas granzyme expression seemed more restricted in the tetramer positive T-cell compartment. In conclusion this study showed that a specific and effective anti-CMV response can be mounted very early after allo-HSCT, even if the donor was seronegative, and gave an insight into the evolution of CMV-specific T cell responses in human, from the onset of reactivation to the stage of chronic infection. and reproducible protein analysis methods that comply with the GLP, GMP and 21CFR Part 11 requirements.Here, LoaC technology can offer a benefit since for protein analysis it integrates sample handling, separation, staining, destaining, detection and digital data analysis. In addition, due to the integration of several individual procedures an increase in throughput and reproducibility can be achieved.We have compared chip-based protein analysis with regard to sensitivity, sizing accuracy and reproducibility to SDS-PAGE. Data were comparable to that obtained from Coomassie-stained PAGE gels. Electropherograms plotting fluorescence intensity against separation time are generated for each sample. Data for individual constituents of a complex mixture are shown along with calculations of concentration and percent total for each protein in the trace.Analysis of 10 samples takes thirty minutes using only four microliters of sample. The data analysis is automatically performed in real-time and is stored and archived in digital format. IQ and OQ/PV tools and services as well as 21CFR Part 11 compliant software tailor the instrument for use in regulated environments. Correlation of Clinical Outcome with Immunophenotype in Islet Transplant Recipients. N. S. Kenyon, 1 X. Xu, 1 D. Han, 1 C. Healy, 2 S. Koester, 2 D. Baidal, 1 C. Ricordi, 1 R. Alejandro. 1 Insulin independence can be achieved via allogeneic islet transplantation in patients with type 1 diabetes. Our aim was to determine the immunophenotype in islet transplant recipients and to determine if alterations in the immune profile (IP) correlated with graft status. Thirteen islet allograft recipients with long-term (N 5 years) type 1 diabetes treated with steroid free immune suppression (rapamycin, FK506 and induction therapy with Zenapax) were monitored serially for changes in IP. All patients experienced insulin independence, with 8/13 eventually returning to reduced dosages of exogenous insulin. Peripheral blood (PB) samples were collected pre and post transplant for 4 color staining and multiparametric analysis of lymphocyte subsets (T, B, NK cell) and activation markers (CD25, CD69, HLA DR). Similar to our observations for cytotoxic lymphocyte gene (CLG) expression in PB, variable changes in IP occurred in the early post-transplant period (with each infusion) which initially did not appear to correlate with graft status. Differences, however, were subsequently observed for both overall white blood cell count (WBC) and IP between stable recipients vs. patients with partial islet allograft loss. In this study, we compared all IP to both CLG data and to results from anti-donor mixed lymphocyte reaction (MLR). Data show WBC decreased to less than 1/2 of baseline in 3/4 stable patients and remained relatively low, but for 7/8 patients that experienced rejection, WBC increased subsequent to elevation of CLG and remained higher. In 3/4 stable recipients, CD3/45 T cells dropped to less than 1/2 baseline and remained at or below this level. Similar to WBC, all T cell counts (including CD3/4 and CD3/8 T cell subsets) dropped initially and then increased after evidence of rejection (indicated by CLG elevation, or onset of hyperglycemia, or initiation of exogenous insulin) in 6/8 patients. Despite anti-IL2R (Zenapax) therapy, 7/8 patients with partial graft loss showed elevation of CD4/25 cells after apparent rejection and 7/8 showed elevation of CD4/69 T cells; 2/4 stable patients showed clear and stable decreases from baseline and the other 2 experienced gradual increases over time. Regarding NK and B cell subsets, trends toward counts that remained below baseline were again seen for stable patients and increases after evidence of rejection were observed in patients with partial graft loss. This suggests that changes in post transplant IP are indicative of alterations in the recipientTs immune response to transplanted islets, as supported by CLG, MLR, and clinical data in stable vs. rejecting patients. We are working to establish flow based methods to assess the functional status of recipient cell in response to both non-specific and donor cell stimulation to ultimately tailor patient therapy.OR-59. K. Gilliam, 1 J. P. Palmer, 1 B. Brooks-Worrell, 1 C. J. Greenbaum, 2 C. Pihoker. 3 1 Medicine, University of Washington, Seattle, WA, USA; 2 Benaroya Research Institute, Seattle, WA, USA; 3 Pediatrics, University of Washington, Seattle, WA, USA.Aims: The incidence of both type 1 (T1D) and type 2 diabetes mellitus (T2D) is increasing in children. Our aim was to examine the relationship between autoimmune measures, HLA, and clinical course. Methods: 28 subjects with atypical T1D (presenting with features such as obesity, acanthosis nigricans, absence of DKA and/or absence of weight loss), and 15 subjects with a clinical diagnosis of T2D were studied at time of diagnosis. Diabetes-associated autoantibodies (DAA) including islet cell, GAD65, IA-2, and insulin autoantibodies were measured. 23 subjects underwent HLA genotyping and were classified as having a high risk (DR4/DQ3; DR3/DQ2), protective (DR15/DQ6), or low risk (other HLA) haplotypes. Subjects were 8-18 years of age at diagnosis, and clinical course was followed in 84% of subjects for a mean period of 47.9 F 18.7 months. Results: Atypical T1D: 25/28 (89%) atypical T1D subjects were positive for at least one DAA. 24/28 (86%) were prescribed insulin at diagnosis and remained on insulin throughout the follow-up period (3 insulin-requiring subjects were lost to follow-up). All 24 insulin-requiring subjects were DAA positive at diagnosis. 2/4 initially non-insulin-requiring subjects remained on oral agents. Both of these were DAA negative, and the one typed subject was HLA low risk. The other 2 subjects initially treated with oral agents subsequently required insulin for glucose control. T2D: 5/15 (33%) T2D subjects were DAA positive at diagnosis. 1/7 HLAtyped subjects had a high risk HLA genotype, 2 had a combination of high risk and protective alleles, and the remaining 4 had low risk or protective alleles. At diagnosis, 14 subjects were treated with oral agents and one with insulin. The subject initially treated with insulin remained on insulin. 7/10 subjects initially treated with oral agents remained on oral agents during follow-up. Three were DAA positive, and one had high risk HLA, but no subject in this group was positive for both DAA and high risk HLA. Two of the three subjects initially treated with oral agents who subsequently required insulin for glucose control were DAA positive, and both had high risk HLA. Conclusions: Children clinically classified with T2D or with an atypical presentation for T1D have a high frequency of autoimmune markers and T1D-associated HLA haplotypes. In addition, these autoimmune markers in children with clinical features of T2D appear to be indicators of a more aggressive diabetes disease process, as has been previously shown in children with typical T1D.OR-60. Development of a Clinical Assay Evaluating Toll-Like Receptor Function. R. P. Deering, J. S. Orange. 1 Immunology, The ChildrenTs Hospital of Philadelphia, Philadelphia, PA, USA.Toll-like receptors (TLR) are transmembrane pattern recognition proteins that participate in innate immune responses. A number of genetic defects influencing the function of these receptors have been identified and are associated with recurrent and/or severe infection. Our goal was to develop a reproducible assay of TLR response for evaluation of TLR function in patients with recurrent infection. Peripheral blood mononuclear cells (PBMC) were isolated and incubated with ligands for TLRs 1/2, 2/6, 3, 4, 5, 6, 7 and 9 . Tumor necrosis factor (TNF) in cell supernatant was then evaluated by enzyme-linked immunosorbent assay (ELISA) as a measure of immune response. Optimal concentrations of, and mean responses to, individual ligands were established in healthy adult control donors. A number of variables were assessed that could affect the assay, including blood anticoagulant, blood storage time, PBMC cryopreservation, assay media, and incubation period. The assay was most reproducible in media containing fetal bovine serum; neither serum-free nor human serum-containing media could be effectively substituted. Cryopreserved PBMCs resulted in a considerably higher TNF production in response to most ligands than freshly isolated cells (31% mean increase amongst all ligands tested). Using optimized assay conditions, three patients with a mutation in the IKBKG gene encoding the NF-kappaB essential modulator (NEMO) protein were ultimately studied as disease controls. TNF responses in patients with IKBKG mutations predicting C417Y, L153R, and exon 9 deletion alterations of NEMO were N= 6.0%, 12.0% and 8.0% of their corresponding controls, respectively. Although a number of variables influence TNF TLR responses this assay can be optimized for clinical use in screening for patients affected by primary immune deficiencies influencing TLR function. Aim of Study: Inhalation of innocuous proteins induces the development of tolerance, an immune response characterized by the development of T reg cells and absence of airway hyperresponsiveness (AHR) or airway inflammation. The reason why some proteins promote allergic sensitization instead of tolerance has been studied extensively but is still controversial. Many allergens possess serine proteinase activity. Some of these allergens/serine proteinases, such as those from house dust mites and cockroachs, can mediate their effects through the Proteinase-Activated . We have previously shown that exogenous PAR-2 activation during allergen challenge enhances allergen-mediated AHR and airway inflammation. Our current hypothesis is that PAR-2 activation at the time of encounter with an inhaled protein may mediate allergic sensitization by preventing the development of tolerogenic responses. Methodology: We used a Balb/c mouse model of tolerance to intranasal (i.n.) administered ovalbumin (OVA). Balb/c mice were administered OVA (100Ag) i.n. Other mice were administered OVA with the PAR-2 activating peptide (PAR-2AP) SLIGRL-NH 2 to mimic inhaled allergens with PAR-2 activating potential. Control mice received OVA with the PAR-2CP (control peptide) LSIGRL-NH 2 or were administered saline alone. All mice subsequently received an interperitoneal (i.p.) immunization with OVA and Al(OH) 3 ten days after the last i.n. Five days following this immunization, mice were euthanized and T cells were isolated from the spleen and cultured in vitro with antigen presenting cells, from a naRve mouse, and OVA for 4 days and proliferation assessed. Other groups of mice were challenged twice with OVA on alternate days, 10 days after the i.p. immunization, and assessed for AHR and eosinophilic inflammation in the lung the day after the last challenge. Results: T cells isolated from mice treated initially with OVA alone or with PAR-2CP proliferated poorly to OVA in vitro, indicating the development of tolerance to OVA while T cells from mice treated with saline alone or OVA and PAR-2AP proliferated vigorously. Furthermore, mice treated initially with saline alone or OVA and PAR-2AP developed AHR and eosinophilic inflammation following OVA challenge while mice treated with OVA alone or with PAR-2CP showed no signs of either. Conclusions: PAR-2 activation in the airways prevents the development of tolerogenic responses towards i.n. administered OVA. These observations indicate that inhaled allergens/serine proteinases may induce allergic sensitization through the activation of PAR-2 in the airways.OR-62. Nasal Vaccination with a Proteosome-Based Adjuvant and Glatiramer Acetate Clears AlzheimerTs B-Amyloid in an Antibody-Independent Fashion.IVX-908 resulted in an 84% reduction of thioflavin S -positive fibrillar amyloid in the hippocampus (pb0.001) and 73% reduction of total brain Ah levels (pb0.001). This effect did not require antibody, as it was observed in B-cell deficient mice. Vaccinated animals developed activated microglia (CD11b+ cells) that co-localized with Ah fibrils, and the extent of microglial activation correlated strongly with the decrease in Ah fibrils. We also noticed a strong correlation between CD11b+ cells and IFN-g secreting cells and increased numbers of T cells (r= 0.9), which may play a role in promoting microglial activation. Our results define an antibody-independent therapeutic approach for the treatment of AlzheimerTs disease, utilizing compounds that have been safely tested or are currently in use in humans.OR-63. Collaboration between Central Tolerance and Peripheral Regulation To Control Autoimmunity.Z. 1 1 Immunology and Immunogenetics, Joslin Diabetes Center/Harvard Medical School, Boston, MA, USA.A variety of mechanisms have been proposed for immunological tolerance of self tissue. Prominent roles have been attributed to central tolerance, illuminated by the role of aire in thymic deletion of autoreactive T lymphocytes, and peripheral regulation, exemplified by the function of Foxp3 in generating CD4 + CD25 + regulatory T cells to suppress pathogenic effectors.Here we report fulminant autoimmunity in very early life and a gravely shortened life-span in mice deficient in both aire and Foxp3 vis-à-vis animals lack either aire or Foxp3. The exacerbated inflammatory damage in the aire and Foxp3 double-deficient animals is particularly prominent in the lung and liver, and also involves several other organs but, despite massive lymphoproliferation, little or no inflammatory infiltration occcurs in many other organs, possibly protected by aire-and Foxp3-indepent mechanism(s). This study highlights the critical importance of both central tolerance and peripheral regulation in maintaining self -tolerance and suggests that there is still more to learn. The eye is an immune privileged tissue that constitutively produces neuropeptides, cytokines, and factors that actively suppress inflammation mediated by innate and adaptive immunity. The neural retina (NR) and retinal pigmented epithelial (RPE) cells are documented to support immune privilege; however, most of our understanding of the mechanisms of ocular immunity has been from analyzing the anterior chamber of the eye. Therefore, to begin to identify factors important in regulating immunity in the retina, we investigated the effects of secreted factors from the NR and the RPE on the activity of resting and activated macrophages. Culture media was placed into the resulting posterior eye cup containing RPE and incubated for 24 hours. These conditioned media (CM) was used to treat resting or endotoxinstimulated macrophages (J774A.1 cell line). After 48 hours, the macrophage supernatants were assayed by multiplex analysis for IL-1beta, IL-6, IL-10, TNF-alpha, GM-CSF. In addition, the RPE-CM and NR-CM were assayed by multiplex analysis. The NR-CM stimulated GM-CSF production by resting macrophages, but was suppressed in endotoxin-stimulated macrophages. Both the RPE-CM and NR-CM suppressed IL-1beta and TNF-alpha production by the endotoxin-stimulated macrophages, while they induced significant levels of IL-10 production by the macrophages. While the NR may support macrophage differentiation through GM-CSF, both the NR and the RPE suppress the production of inflammatory cytokines (IL-1beta and TNF-alpha) by endotoxin-stimulated macrophages, while causing the same macrophages to produce the anti-inflammatory cytokine, IL-10. Such results suggest that the mechanisms of immunosuppression by the retina may involve factors that suppress classical activation while promoting alternative activation of macrophages. Therefore, both the neuroretina and RPE contribute to the mechanisms of retinal immune privilege.Supported by grants from the USAMRMC.OR-65. Treatment with a Donor-Specific Transfusion and Anti-CD154 mAb Induces Non-Responsiveness in a Population of T Cells That Recognize Alloantigen Via Indirect Antigen Presentation.N. E. Phillips, 1 D. L. Greiner, 1 J. P. Mordes, 1 A. 1 1 Medicine/Diabetes, University of Massachusetts Medical School, Worcester, MA, Viet Nam. Treatment with a donor-specific transfusion (DST) and anti-CD154 monoclonal antibody (mAb) induces prolonged allograft survival in mice, in part by deleting host CD8 + T cells that recognize alloantigen by direct antigen presentation. The fate of host T cells that recognize alloantigen via indirect antigen presentation in mice treated with costimulation blockade is unclear. In this study, we investigated the fate of Tg361 TCR transgenic CD4 + and CD8 + T cells that recognize alloantigen via indirect antigen presentation. Using CFSE-labeling, we first document that both Tg361 transgenic CD4 + and CD8 + T cells proliferate in vitro and in vivo in response to allo-stimulation. Treatment of mice circulating Tg361 T cells with DST plus anti-CD154 mAb, however, fails to delete these CD4 + and CD8 + alloreactive T cells, and instead renders them non-responsive to re-challenge with alloantigen. Mice circulating these nonresponsive alloreactive T cells also fail to reject skin allografts. The non-responsive state of Tg361 T cells is not reversed by the addition of IL-2, anti-CD28 mAb, or an agonistic anti-CD134 mAb in the presence of antigen, protocols that have been successful in reversing both the clonal and adaptive anergic states of tolerized CD4 + cells. The non-responsive CD4 + and CD8 + alloreactive T cells arecapable of activation, however, as evidenced by their robust in vitro proliferation in response to anti-CD3 mAb in the presence of costimulation. These data document a non-deletional mechanism by which costimulation blockade can block host alloreactive T cell responses and prolong graft survival. Successful Induction of Mixed Chimerism and Tolerance with Non-Myeloablative Conditioning in Mice Presensitized with Fully Mismatched Skin Grafts. have used B cell deficient mice to evaluate the ability to tolerize presensitized T cells via mixed chimerism induction through bone marrow transplantation (BMT). B cell deficient B6-AMT and wildtype B6 (H-2 b ) mice, presensitized with B10.A (H-2 a ) tail skin, received non-myeloablative conditioning (anti-CD4, CD8, NK, CD40L & OX40L monoclonal antibodies and 3 Gy total body irradiation) before BMT with 80Â10 6 B10.A bone marrow cells (BMC) . Twelve weeks after rejection, the presence of anti-donor IgG in the serum of the presensitized B6 mice was confirmed by indirect staining and flow cytometry. Multi-lineage chimerism was monitored at 2, 4, 6, and 12 weeks post-BMT by flow cytometry. Thus, anti-donor IgG antibodies present in wild-type mice after presensitization posed a strong barrier to donor marrow engraftment. In contrast, 80% of the AMT mice showed engraftment of donor BMC with stable long-term multi-lineage donor chimerism. Importantly, chimeric AMT mice accepted donor grafts long-term, while third party grafts were rejected by 2 weeks. Thus our results show that pre-existing T cell immunity to BMC donor antigens may be overcome by non-myeloablative mixed chimerism induction in presensitized mice. We have previously shown that cellular necrosis augments CD40-mediated interleukin-12 secretion by human monocytederived dendritic cells. In the present study, we compare the dendritic cell response to cellular necrosis in atopic individuals with non-atopic control subjects. Using an in vitro culture system, monocyte-derived dendritic cells were stimulated with either necrotic K562 cells or a combination of TNF-alpha and IL-1beta. We demonstrate that dendritic cells from atopic individuals secreted significantly less interleukin-12p40 in response to necrotic cell products compared with dendritic cells from non-atopic subjects. Upon stimulation with necrotic cells and CD40 crosslinking, dendritic cells from atopic subjects secreted significantly less interleukin-12p70. Furthermore, CD80, but not CD86, was upregulated by necrosis significantly less on dendritic cells of atopic individuals compared with normal subjects. In contrast, the response of dendritic cells from atopic subjects to TNF-alpha and IL-1beta was not significantly different from normal individuals. We conclude that atopy is associated with a defective response of dendritic cells to necrotic cell death, which may play a role in the mechanism of atopic sensitisation. CD150 (SLAM) Modulates TLR and CD40 Pathways in Monocyte-Derived Dendritic Cells.B. Rethi, 1,3 P. Gogolak, 1 E. Rajnavolgyi, 1 C. Terhorst, 2 A. Lanyi. 1 1 Institute of Immunology, University of Debrecen Medical and Health Science Center, Debrecen, Hungary; 2 Division of Immunology, Beth Israel Deaconess Medical Center, Boston, MA, USA; 3 Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm, Sweden. SLAM (CD150, Signaling Lymphocyte Activation Molecule) is a self-ligand receptor on the surface of activated T-and Blymphocytes, macrophages and dendritic cells (DC) . Its importance at the interface of adaptive and innate immune responses is underscored by SLAM being a receptor for Measles virus, which induces immunosuppression. Moreover, recent reports indicated that high expression levels of SLAM in T-lymphocytes of patients infected with M. tuberculosis or M. leprae were associated with milder pathology.As the function of SLAM on the surface of human dendritic cells is poorly understood, we examined the effect of SLAM/ SLAM interactions on activation signals in human peripheral blood monocyte-derived dendritic cells. The effect of SLAM on CD40L-induced DC activation was analyzed in co-cultures of DC with L929 cells expressing CD40L alone or in combination with SLAM. CD40L-induced IL-12 production was strongly inhibited by SLAM engagement and resulted in a DC phenotype that was less potent to induce differentiation of naive T lymphocytes into IFN-g producing Th1 effector cells. Interestingly, the ability of these bSLAM-educatedQ DC to support the proliferation of naive T cells was significantly increased. To determine the effect of SLAM on different TLRinduced DC activation, L929 cells or L929 cells expressing SLAM were co-cultured with immature dendritic cells in the presence of LPS or poly-IC. In DC activated by CD40L and LPS, SLAM engagement reduced IL-12 production to the level of cultures activated by LPS and SLAM, indicating that SLAM modulates these pathways independently.Thus, our findings suggest a dual function for SLAM in monocyte-derived DC that allows SLAM to exert opposing effects on IL-12-dependent functions, based on the received activation signals.OR-69. Activation of Human NK Cells by Plasmacytoid DC and Its Modulation by CD4 + T Cells and CD25 hi T Regulatory Cells.C. Romagnani, 1,2 M. Della Chiesa, 2 S. Kohler, 1 L. Moretta, 2, 3 A. Moretta, 2 A. Thiel. 1 1 Clinical Immunology, German Rheumatism Research Center, DRFZ, Berlin, Germany; 2 Dipartimento di Medicina Sperimentale, Universita di Genova, Genova, Italy; 3 Istituto G. Gaslini, Genova, Italy.Background: Plasmacytoid dendritic cells (pDC) represent a specialized cell population that produce type I interferon in response to virus. However, although pDC-derived type I interferon is a potent modulator of NK cell functions and NK cells are essential for antiviral immunity, the role of pDCs in coordinating NK cell functions has not yet been elucidated in detail, especially in humans.Objective of the study: to investigate the interplay between human pDC and NK cells and to evaluate how CD4 + Th and CD25 hi T regulatory (Treg) cells can modulate these interactions.Methods: Highly purified FACS-sorted human pDC, NK cells, CD4 + CD25 hi Treg cells and CD4 + CD25 neg T cells were cocultured and NK cells were analysed for CD69 expression, for proliferation after staining with CFSE, for cytokine production using Bioplex and for cytotoxicity in 4 h-51 Cr release assay. pDC were activated with IL-3 and CpG-A ODN2216 before co-culture with NK cells.Results: pDC, following engagement of TLR9, can activate autologous NK cells, as indicated by the induction of surface CD69 expression. Under these conditions, pDC can also enhance NK cell effector functions, including cytotoxicity and cytokine production. Moreover, they can induce proliferation of CD56 bright CD16-, but not of CD56 dim CD16 + NK cells. This activity can be highly up-regulated in an IL-2-dependent fashion by autologous CD4 + CD25-T cells. Strikingly, CD4 + CD25 hi Treg cells can inhibit proliferation of NK cells induced by the interplay of pDC and T helper cell, while they do not influence NK cell activation or proliferation induced by pDC alone.Conclusions: This is the first demonstration in humans that pDC can activate NK cells, enhance their effector functions and induce proliferation. In addition, it is the first demonstration that CD4 + Th and CD25 hi Treg cells can modulate NK cell proliferation, implying a direct role of adaptive immune response in amplifying or inhibiting innate immunity.OR-70. Hepatitis C Virus Core Protein Induced, Monocyte-Mediated Mechanisms of Reduced IFNA and Plasmacytoid Dendritic Cells Loss in Patients with Chronic HCV Infection.A. 1 1 Medicine, University of Massachusetts Medical School, Worcester, MA, USA.Immune responses to acute hepatitis C virus infection are insufficient in most individuals leading to chronic infection in the majority of infected individuals. Current successful therapies of HCV infection are based on use of IFNa. Type I interferons (IFN), including IFN-alpha (IFNa), inhibit viral replication and promote antiviral immune responses. Limited and controversial information is available related to the capacity HCV-infected patients to produce endogenous IFNa.The purpose of this study was to investigate the functional capacity of plasmacytoid dendritic cells (PDCs), the main producers of IFNa, in patients with chronic HCV infections compared to controls. We found significantly decreased production of IFNa, determined by ELISA, in PBMCs of HCV patients upon stimulation with PDC-specific TLR9 ligands, CpG-A DNA (pb0.003) and HSV KOS (pb0.004) . This correlated with a decreased frequency of circulating PDCs (determined by flow cytometry as lineage-/CD4+ or BDCA2+/ CD123+) in HCV patients compared to controls (HCV 0.11 F 0.09%, control 0.25 F 0.15%; pb0.003). PDCs purified from HCV patients produced lower levels of IFNa compared to controls (pb0.009) and were apoptotic, as determined by staining with Annexin V and DNA laddering. In vitro stimulation with CpG-A DNA or HSV KOS lead to increased cell death in PDCs from HCV patients compared to controls (pb0.01). There was no correlation between the loss of PDCs and HCV viral count, genotype, or liver functions. Importantly, there was no reduction in PDC frequency or IFNa production in patients with sustained virological response after HCV elimination therapy suggesting a role for viral derived mechanisms for the DC defects. We found that recombinant HCV core protein did not directly affect PDC functions, but it significantly reduced TLR9-triggered IFNa production in PBMCs (pb0.001). We determined that HCV core protein activated monocytes to produce IL-10 and TNFa. Anti-IL-10 and anti-TNFa neutralizing antibodies were additive in restoration of IFNa production in TLR9+HCV core stimulated PBMCs. Depletion of monocytes from PBMCs abolished IL-10 and TNFa production and prevented HCV core-induced inhibition of PDC IFNa production.Our results show that HCV core protein modulates hostTs ability to produce IFNa by indirectly interfering with PDC function via monocyte-derived cytokines. We reveal that the viral-induced mechanisms of PDC loss and IFNa production defects are likely to contribute to chronic viral persistence and may provide mechanistic explanation for the therapeutic benefits of IFNa therapy in HCV infection.OR-71. Delayed IL-10 Induced Human Tolerogenic DC Inhibit Naive T Cell Proliferation by Mechanisms Other Than Their Exaggerated PD-L1/2 Induction.F. Li, 1 K. Laudanski, 1 L. Perez, 1 C. L. Miller-Graziano. 1 1 Surgery, University of Rochester Medical Center, Rochester, NY, USA.Previous data indicated that IL-10 treated human monocytes (MO) differentiate to macrophage (Mac) expressing elevated Mac markers like MRP8/4 while addition of IL-10 to monocytes after their partial differentiation to dendritic cells (MO IL-4 & GM-CSF 3 days then IL-10 added for additional 2 days) induces a tolerogenic dendritic cell (tol DC) which can diminish T cell activation. The mechanisms for tolerogenic DC inhibition of T cells is still undefined, but is suggested as related to upregulation of inhibitory costimulation ligand-receptor combinations. Here, tol DC were generated from 7 control donorsT MO by adding IL-10 after 3 days of culture of MO with IL-4 & GM-CSF and then inducing for an additional 2 days. Addition of IL-10 to the DC differentiation cultures did not significantly decrease DC CD1a levels (68 F 15% positive) versus those of IL-4+GM-CSF classic DC controls (76 F 14% positive), or their CD209 levels (97 F 1 versus 98 F 1% positive) nor increase Mac characteristics (CD14 5 F 2 versus 3 F 4% positive, or MRP8/14 b1% positive expression). However, tol DC expression of the inhibitory costimulatory ligand PD-L1 but not PD-L2 was significantly (b0.0001) increased from the 13 F 5% of classic DC to 36 F 9% positive. We have previously shown that tol DC are unable to act as adequate antigen presenting cells (APC). To assess the inhibitory activity of these tol DC, they were added to autologous T cell cultures in the presence of immobilized anti CD3 plus CD28 (iCD3+CD28). Since iCD3+CD28 stimulate T cell proliferation in the absence of any APC, any diminished proliferation in the presence of tol DC would indicate a T cell inhibitory rather diminished APC effect. The addition of 2Â10 4 to1 DC significantly (P = 0.035) reduced T cell proliferation to anti iCD3+CD28 as compared to T cell cultures with classic or no DC. However, there was no correlation between the degree of increased tol DC PD-L1 expression and then mediation of decreased T cells proliferation. Tol DC with the highest increases in PD-L1 did not exhibit the highest inhibitory activity. These data suggest that the tol DC function of reducing autologous naive T cell proliferation to T cell receptor stimulation resulted from induction of other inhibitory costimulatory receptors (ILT3/4) rather than the induction of PD-L1 or PD-L2. Nevertheless, the IL-10 induced augmentation of DC PD-L1 expression may still contribute to T cell inhibition and anergy induction when tol DC interact with previously activated T cells whose upregulated PD-1 levels can be triggered by exaggerated tol DC PD-L1 levels.OR-72. Induction of Heme Oxygenase-1 (HO-1) Inhibits Dendritic Cell Differentiation and Adaptive Immunity.J. J. Listopad, 1 T. Ritter, 2 R. Sabat, 1 K. Asadullah, 1 W.D. 1 1 CRBA Dermatology, Schering AG, Berlin, Germany; 2 Institute of Medical Immunology, Charite, Humboldt University, Berlin, Germany.The strong immunosuppressive potency of the stress protein HO-1 has been proven in several models of autoimmunity and transplantation. The underlying immune mechanisms, however, are poorly characterized. In our study, the potent HO-1 inducer Cobalt Protoporphyrine IX (Co-PPIX) strongly suppressed T cell proliferation in mixed lymphocyte reaction (MLR) . As possible mechanism we demonstrated a selective Co-PPIX induced increase of HO-1 expression in monocytes associated with depression of accessory molecule expression and stimulatory cytokine secretion. The likewise induction of HO-1 in monocytederived dendritic cells (MDDC) by Co-PPPIX was associated with an almost complete inhibition of the differentiation, maturation, and function of MDDC. So, a strong decrease of the expression of DC markers (CD1a, CD83) and accessory molecules (HLA-DR, CD86) was observed. Whereas IL-12 secretion was inhibited, IL-10 production increased. The antigen-presenting capacity of Co-PPIX treated MDDC was strongly diminished in lymphocyte transformation assay and MLR. Together these changes indicated a switch of the DCs to an immature, nonstimulatory phenotype. In vivo, Co-PPIX treatment before challenge dose-dependently depressed ear inflammation in DNFB (Type 1) and TMA (Type 2) induced contact dermatitis in mice. Remarkably, Co-PPIX even more strongly inhibited T-cell-dependent inflammation when applied around sensitization. We hypothesize that the inhibition of DC differentiation, maturation, and function is a crucial mechanism for the suppression of adaptive immunity by HO-1 induction in vitro and in vivo.OR-73. Donor-Specific Allograft Tolerance by Administration of Recipient-Derived Immature Dendritic Cells and Suboptimal Immunosuppression.G. Beriou, 1 H. Peche, 1 C. Guillonneau, 1 E. Merieau, 1 M. C. Cuturi. 1 1 U643, INSERM, Nantes, France. The benefits of allogeneic immature bone marrowderived dendritic cells (iBMDCs) on allograft survival have been reported in several studies. However, in contrast to protocols based on the injection of donor-derived DCs, the administration of recipient-derived DCs would be much more applicable to cadaveric organ transplantation. We recently showed that injection of recipient-type iBMDCs the day before transplantation induced a significant prolongation of allograft survival. In the present study, we aimed at improving this protocol to induce allograft tolerance.Methods. iBMDCs were generated from LEW.1A rat bone marrow precursors with low-dose GM-CSF and IL-4. After 8 days of culture, adherent cells displayed immature phenotype, characterized by low MHCII and CD86 expression. Various amounts of iBMDCs (3 to 15Â10 6 cells) were administered i.v. to syngeneic LEW.1A rats before and after transplantation of an allogeneic LEW.1W heart, with or without additional suboptimal immunosuppression, consisting of Rapamycin (0.4mg/kg/day, d0-d14, orally) or LF15-0195, a new deoxyspergulalin analog (1.5mg/kg/day, d0-d9, i.p.) Interestingly, combining injection of iBMDCs and LF15-0195 had striking synergistic effect, and induced definitive allograft acceptance in 92% of recipients. Tolerant iBMDC-LF15-0195 treated recipients accepted donortype, but not third party-type skin grafts, demonstrating that tolerance was donor-specific. We hypothesized that under LF15-0195 treatment, iBMDCs could maintain their immature phenotype and function. The effects of LF15-0195 treatment on the in vivo maturation of the administered iBMDCs are currently under investigation.Conclusions. Thus, we demonstrated that donor-specific allograft tolerance can be induced by a single injection of syngeneic iBMDCs one day prior to transplantation, and a suboptimal immunosuppressive treatment with LF15-0195. The reported findings may contribute to the development of new therapeutic strategies to induce transplantation tolerance in clinical settings. Among DC subsets, the reconstitution of the natural type Iinterferon-producing PDCs has been proposed to play a major role in establishing immune competence. Therefore, we investigated the impact of circulating PDCs measured at the 3rd month after reduced intensity conditioning (RIC, fludarabine-based conditioning) allo-SCT, in 54 patients with hematological and non-hematological malignancies who received a RIC-allo-SCT from an HLA-identical sibling, in order to determine whether this could provide an indicator for long term outcome. In a multiple logistic regression analysis including all relevant parameters (demographic and graft characteristics, RIC regimens, CMV infections, and acute GVHD), only the absence of grade II-IV acute GVHD was associated with an improved PDC recovery at 3 months (P = 0.003; OR=6.4; 95%CI, 1.9-22). Being the major type I IFNsecreting cells, we also investigated whether PDCs recovered after allo-SCT are functional in response to viral stimulation. Patients experiencing grade 0-I aGVHD could secrete significantly higher amounts of IFN-alpha as compared to patients with grade II-IV aGVHD (mean, 91 vs. 0 pg/ml respectively; P = 0.002), likely highlighting the deleterious impact of corticosteroids therapy on PDC function. The CD34+ stem cell dose and other lymphoid subsets infused with the allograft did not affect PDC recovery. Though PDC count could not predict death from progression or relapse, patients with a bhighQ PDC recovery profile had an improved overall survival (OS; P = 0.03), in contrast to patients with a blowQ PDC recovery profile who had an increased incidence of late transplant-related mortality (GVHD, infections) (P = 0.03). In addition, the overall incidence of late infections (viral, fungal and bacterial) was significantly higher in the blowQ PDC recovery group as compared to the bhighQ PDC recovery group (59% vs. 19%; P = 0.002), illustrating the importance of PDCs in antiinfectious immune responses. The role of rare immune effector cells would tend to be more evident in truly RIC and less toxic regimens. In this study, we could show that monitoring of PDCs may be useful for patientsT management (closer surveillance, infection prophylaxis. Severe combined immunodeficiency syndromes (SCIDs) are characterised by absent T-and B-lymphocytes function. SCIDs are usually fatal early in infancy without successful immune reconstitution. We previously reported on a patient affected with an Xlinked SCID, who received a late Bone Marrow Transplantation at 5,2 years of age. During the follow-up a short stature due to peripheral Growth Hormone (GH) hyporesponsiveness and abnormalities of the protein phosphorylation events following GH receptor (GHR) stimulation were observed. Mutational screening and expressional analysis failed to reveal any molecular alteration of GHR, JAK2 and STAT5a/b genes. Since we hypothesized a role for the g chain in GHR signaling, in this study we evaluate GHR/g element functional interaction in EBV transformed lymphocytes (BCLs) from X-SCID PTs and CTRs. The functional response to GH, the pattern of GHR induced Ptyr and STAT5 nucleus translocation were studied. GH enhanced proliferation of CTRs BCLs in a dose-dependent fashion, with a maximal effect at 200 ng/ml. In contrast, PTs cells did not proliferate at all. Cytofluorimetric analysis did not reveal any difference in GHR expression. In PTsT cells, GH stimulation failed to induce phosphorylation of proteins of 90-92 kDa corresponding to STATs molecules in contrast to what observed in CTR, in which a peak of STAT5 phosphorylation between 5 and 15 min was observed. In all cell lines examined, STAT5a and b protein expression was comparable. In addition, in CTR cells GH induced nuclear translocation of STAT5 evaluated through confocal microscopy; in contrast, in PTs cells no efficient translocation occurred after GH stimulation. Here we report a previously unappreciated functional interaction between gc and GHR, which eventually leads to the activation and intranuclear translocation of STAT5 protein.OR-76. Novel Humoral Immunodeficiency in Humans Associated with Deleterious Homozygous Mutation in CD19.D. Castano, 1 P. J. Patino, 1 C. Woellner, 2 U. Salzer, 2 B. Grimbacher, 2 C. J. Montoya, 1 J. C. Orrego, 1 C. Rugeles, 1 J. L. Franco. 1 1 Group of Primary Immunodeficiencies, SIU-University of Antioquia, Medellin, Colombia; 2 Clinical Immunology and Rheumatology, University of Freiburg, Freiburg, Germany.In B cells, CD19 is found in a complex with the complement receptor CD21, the tetraspan membrane protein CD81, and CD225, and is critical both to balance antigen-induced BCR-mediated signaling thresholds in B cells and to functionally link CD21 with the BCR following co-recognition of C3d-bearing Ag. CD19 is a 95 kd transmembrane protein with two extracellular Ig domains and a cytoplasmic tail containing several tyrosine residues that become phosphorylated after cross-linking of the BCR, allowing the interaction with SH2-containing cytoplasmic proteins and linking CD19 to downstream signaling cascades. In mice, mutations in CD19 lead to hypogammaglobulinemia, impaired Bcell memory, low CD5+/B1 B cells and decreased germinal center formation. Here we present three adult siblings (one male and two females) affected with recurrent respiratory and gastrointestinal tract infections since childhood and low serum IgG, IgA and IgM. Peripheral blood CD20+CD22+ B cells were within normal ranges by FACS, but showed profoundly reduced surface expression of CD19 in all three patients. DNA sequencing of CD19 revealed a homozygous deletion of two nucleotides in exon 11 (c.1428delAG), leading to a frameshift and a premature STOP codon in all patients. Six relatives in the family were heterozygous. B-cell subpopulations in PBL showed significant decreases in isotype-switched memory cells (CD27+IgD-) and low CD5+ cells in all patients. Isohemagglutinins where decreased while soluble CD21 levels were slightly increased in all patients. TonsilTs morphology and cellularity in one CD19-deficient patient were normal (CD3, CD79a, CD20, CD5, Bcl-2, and Ki67) with highly active lymphoid follicles. Mutations in TACI Are Associated with Immunodeficient Phenotypes in Humans.affected individuals. The mutation in the first family affected a highly conserved cysteine residue (C104R) in the extracellular domain of TACI. The mutation observed in the second family was a nonsense mutation at position 144 (S144X) leading to a putative truncated TACI protein consisting only of its extracellular domain. We then extended our screening to patients with sporadic CVID and found 11 out of 139 patients, who carried a heterozygous mutation. Two of these affected the conserved cysteine residue (C104R), seven were located within the transmembrane region (A181E) and two were in the intracellular part of the protein (S194X and R202H). FACS staining of patientś B cells with heterozygous mutations revealed normal TACI surface staining. After stimulation with common mitogens (IgM, Il-2, CD40, IL-4) B cells from patients with mutations in TACI proliferated normally. A tonsil biopsy in one of the patients revealed prominent enlarged germinal centers with hypercellularity of B cells. Clinically the patients presented with hypogammagloblobulinemia, especially low IgM, and displayed signs of lymphoproliferation and autoimmunity at a very high frequency.Conclusions: In our evaluation of TACI as a candidate gene in patients with CVID we found both homozygous and heterozygous mutations in familial and sporadic CVID cases. Three mutations lead to substitution of highly conserved amino acids and two are nonsense mutations. The human TACI-deficient phenotype consists primarily of a humoral immunodeficiency and thus differs from the murine model, however, signs of autoimmunity and lymphoproliferation are also evident. Maternal engrafted T cells occur in patients with SCID to a variable extent often with absence of clinical signs of GVHD. In this report we describe maternally engrafted TCRgy cells in two children (RK & JR) with Artemis and common chain deficiencies respectively. The purpose of this study was to characterise these populations in detail. Peripheral blood from both children was phenotyped and functionally characterised by response to mitogens. Both children were delivered at term after a normal pregnancy and were vaccinated according to protocol; in addition RK received BCG at birth. Both presented at 5 months with PCP, low serum immunoglobulins, lymphopenia and failure of lymphocytes to respond to mitogens. RK presented with a T lymphocyte count of 1.1 Â 10 9 /l positive for TCRgy, CD3, CD8 and CD45 R0. No B cells were found. JR presented with a T lymphocyte count of 1.1 Â 10 9 /l positive for TCRgy, CD3, CD4, CD8, CD45R0 and CD45RA. B cells numbered 0.6 Â 10 9 /l. DNA was isolated from peripheral blood cells and amplified with Biomed primers to TCRg and TCRy. In both children clonal TCRg and TCRy rearrangements were found compatible with TCRgy cells utilising TCR Vg-Jg1.3/2.3 and TCR Vy1-Jy1, TCR Vg-Jg1.2 and TCR Vy genes respectively. We present evidence in two children with significantly raised TCRgy populations of maternal origin in blood. While neither of the children had clinical evidence for GVHD, RK who received BCG at birth experienced BCG lymphadenitis, skin and gut biopsies from this child were found to have infiltrates of TCRgy cells that were shown to be clonally identical to the TCRgy cells in blood. In this case it is possible that the clone arose in direct response to BCG vaccination at birth, however, cells were not investigated from mother for their response to BCG. These data confirm a previous report where clonal TCRgy cells were found to cross the placenta but failed to initiate GVHD in the neonate. In this child with SCID clonal TCRgy cells were also shown to be present in the mother. In this abstract the origin of these cells was not resolved. T cell receptor variable beta chain (TCRBV) PCR is frequently used to investigate TCRBV usage in autoimmune diseases, infection and cancer. Because the TCRBV locus contains more than 50 variable regions, a large number of forward primers has to be used to cover all TCRBV segments. Previous studies simplified the TCRBV analysis by performing a multiplex PCR with 26 primers divided into 5 groups. While this approach has been worked out for single cell rtPCR, it is very time consuming and costly and can hardly be applied to large scale screening. To further simplify TCRBV analysis, we established a new semi-nested rtPCR method with two sets of degenerate primers covering 85% and 15% of the TCRBV genes respectively. For single cell analysis, we extended the rtPCR by designing a nested primers located in the TCRBV constant region. The specificity of the primes was confirmed by screening cDNAs from more than 200 T cell clones which were previously defined for their TCRBV usage by flow cytometry and conventional TCRBV rtPCR. We retrieved all TCRBV gene segments comprised in the sample with our primer sets. High sensitivity was demonstrated by successful amplification of rearranged TCRBV genes of single T cells sorted from body fluids or dissected from tissue. This new approach allows fast and cost-effective high throughput analysis of T cell receptor rearrangement at the single cell level facilitating studies on T cell responses in human diseases.Single T cells were directly cloned with mitogen into 96 well plates from pancreatic draining lymph nodes (PLN) from human subjects with Type 1 diabetes and control subjects, with the generation of over 515 independent T cells clones. We sequenced the T cell receptor alpha and beta chains from these clones: modest or no expansion of TCR chains was seen in PLN from non-diseased subjects, including a subject with Type 2 diabetes, while a high degree of T cell expansion was observed in longterm diabetics, but not a recent onset diabetic with CD4+ T cells infiltrating the islets. Using a candidate antigen screening approach and EBV transformed B cells as antigen presenting cells, clonally expanded T cell clones from PLN from both longterm diabetics responded to insulin A chain 1-15 in the context of DRB1*0401 and not in the context of DRB1*0301 or to other insulin, GAD65, or MBP85-99 peptides and was specifically blocked by anti-DR antibody. T cell clones from the PLN of a DR4+ normal, from the cerebrospinal fluid of a DR4+ multiple sclerosis patient, a MBP85-99 reactive T cell clone from the periphery of a multiple sclerosis patient, and non-expanded T cells from one of the long term diabetic DR4+ PLN did not recognize the insulin peptide in the context of DRB1*0401. Our results are the first to establish T cell clonal expansion in human pancreatic draining lymph nodes from subjects with Type 1 diabetes with the identification of a cognate autoantigen. These experiments demonstrate the feasibility of non-biased T cell cloning from the draining lymph node of an organ targeted in an autoimmune disease to identify a putative autoantigen. Moreover, these results confirm in humans results in the NOD mouse model identifying insulin as a critical autoantigen in diabetes. B7/CTLA-4 interactions negatively regulate T cell responses and are necessary for transplant tolerance induction. Tolerance induction may therefore be facilitated by selectively inhibiting the B7/CD28 pathway without blocking that of B7/CTLA-4. In this study, we selectively inhibited CD28/B7 interactions using the JJ319 modulating anti-CD28 monoclonal antibody in the LEW.1W to LEW.1A rat model of acute kidney graft rejection. On the long term (N100 to 300 days), kidney graft function was normal and stable in tolerant recipients with an absence of histological lesions of chronic rejection. Tolerant recipients developed alloantibodies of the Th2type against donor MHC class II molecules, unlike untreated rejecting controls that developed Th1-type antibodies against MHC class I and II molecules. PBMC and spleen cells from tolerant animals did not proliferate against donor cells in MLR but proliferated against third party cells. However, purified T cells were fully reactive suggesting a regulation of T cells by a non-T cell population. The depletion from PBMC of either CD80 or CD86-positive, non-T cells, fully restored this reactivity whereas the depletion of B cells, CD11b/c + , MHC II + and CD8 + cells had no effect. Over represented NK cells expressing CD80/86 were found partially responsible for this suppressive effect. In vivo, pharmacologic inhibition of these enzymes led to the rejection of the otherwise tolerated transplants. This study demonstrates that an initial selective blockade of CD28 generates B7 + non-T regulatory cells and a kidney transplant tolerance sustained by the activity of IDO and iNOS. In contrast, granulomas were not detectable in IL-4-deficient TCRa double knockout (aIL4 DKO) and B cell-deficient TCRa (aA) DKO mice. Colitis in TCRa KO mice is characterized by a marked increase of IL-4 production, whereas aAIL4 TKO mice showed significant increase in IL-12p40 and IFN-g production. Interestingly, the development of granulomatous colitis was associated with an increase of immature myeloid dendritic cells (DCs) as indicated by the presence of DCs with CD86-CD11b + CD11c + phenotype. This was associated with a marked increase in IL-17 expression by CD4 + T cells in aAIL4 TKO mice, compared to other KO mice. The IL-12p40/p19 production by colonic LP DCs was further upregulated by a toll-like receptor 9 ligand (CpG) and significantly downregulated by IL-4 as well as IgG (Fc fraction). To test the ability of immature myeloid DCs to induce granulomas, purified immature myeloid DCs isolated from the granulomas of aAIL4 TKO mice (6 months of age) or bone marrow-derived immature or mature myeloid DCs from WT mice were directly injected into the ileocecal valve of recipient young aAIL4 TKO mice (9 weeks of age) following laparotomy. Importantly, the transfer of colonic myeloid DCs as well as also bone marrow-derived WT immature myeloid DCs led to the development of granulomas in the recipient aAIL4 TKO mice. In contrast, the transfer of mature myeloid DCs failed to do so. These findings indicate that immature myeloid DCs have the ability to induce granulomas under specific intestinal inflammatory conditions characterized by Th1 dominated immune responses (or absence of Th2 environment) and impaired B cell functions. Multiple sclerosis (MS) is a demyelinating disease of central nervous system (CNS) characterized by plaques of infiltrating CD4+ and CD8+ T cells. Although EAE, an animal model for MS, is considered a CD4+ T cell mediated disease, recent reports have suggested that myelin-specific CD8+ T cells can also cause EAE in susceptible strains of mice. However role of HLA class I in pathogenesis of MS is not well defined due to lack of proper animal model. In this study, we have generated HLA class I transgenic mice to investigate the function of these molecules in disease pathogenesis of EAE. Transgene (HLA-A11) were introduced into class I deficient mice by breeding to eliminate the effects of endogenous class I molecules. T cells from the A11 tg mice responded to PLP 41-60 peptide in a dose dependent manner but no response was seen in KboDbo mice expressing same mouse class II. Further, using in vitro antibody blocking experiments, the T cell response in tg mice was shown to be mediated by both CD4+ T cells as well as CD8+ T cells and restricted by the HLA class I transgene molecule. PLP 41-60 peptide also induced pronounced neurological disease in A11 tg mice characterized by brain ataxia, spinning, spastic reflexes and head tilt. These mice also showed CNS pathology consisting of heavy inflammation in menengial and cerebellum region of brain. This is the first animal model describing a encephalitogenic role of HLA class I-restricted CD8+ T cells. Further study is underway to understand role of HLA class I molecule in predisposition and onset of EAE. USA; 2 Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, CA, USA; 3 Palo Alto VA Health Care System, Palo Alto, CA, USA; 4 Pathology, Stanford University School of Medicine, Stanford, CA, USA. Costimulation and Tolerance The pathogenesis of rheumatoid arthritis (RA) involves the deposition and excessive local generation of fibrin in the inflammed joint. It is belived that the imbalance of fibrinolysis and coagulation within the rheumatoid joint differentiates RA from other joint diseases. In this model we demonstrate strong T cell reactivity to fibrinogen with no cross-reactivity to collagen type II. Using proteome microarrays containing proteins and peptides representing the putative autoantigens in RA, we also find strong B cell reactivity to fibrinogen, and robust autoreactive B cell spreading to collagen types I, II and V, human cartilage glycoprotein 39, and citrulline-substituted peptides derived from filaggrin. We also show that arthritis can be adoptively transferred to naive mice with either sera or whole splenocytes from diseased mice. Clinical symptoms from both immunized and adoptively transferred mice include erythema and mild swelling that encompass the ankle, foot, and digits. Histopathological analysis of H&E stained joint sections indicate a mononuclear infiltrate within the inflamed synovial membrane. This new fibrinogen-induced arthritis mouse model may provide additional insight into understanding the disease mechanisms and developing novel therpeutic interventions for rheumatoid arthritis. Materials and methods: Groups of 8-12 week old C57BL/6 mice transgenic for the extracellular domains of HLA-DR4 were immunized subcutaneously with 50 ug of a maltose binding fusion protein for U1-70kD ribonucleoprotein (70K-FP) in 50 ul of either U1-RNA in PBS (at 1 ug/ul) or CFA. Control groups also included mice injected with 50 ug of maltose binding protein lacking the 70K-FP with U1-RNA or CFA. Sera from mice were examined for autoantibodies using immunoblot and ELISA. Tissues were obtained at necropsy, stained with hemotoxylin and eosin, and examined in a blinded fashion.Results: Anti-U1-70kD and other anti-ribonucleoprotein (RNP) antibodies developed both in mice immunized with 70K-FP + CFA and 70K-FP + U1-RNA. MCTD-like interstitial and perivascular lung disease developed in groups of mice immunized with either 70K-FP + CFA (60%) or 70K-FP + U1 RNA (75%). Anti-RNP antibodies and lung disease was not observed in control mice. Injection of a single dose of U1-70kD RNP with its physiologically associated U1-RNA was adequate to induce autoimmunity in mice transgenic for the HLA-DR4 allele associated with susceptibility to MCTD.Conclusions: A single injection of HLA-DR4 transgenic mice with 70K-FP+ U1-RNAor70K-FP+ CFA inducedanti-RNP autoimmunity and interstital lung disease. Proteoglycan-Induced Arthritis: A New and Unique TCR Transgenic Arthritis Model.To characterize pathogenic effector T cells in arthritic mice, and to map T cell recognition sites in human and mouse cartilage proteoglycan (PG), we have generated PG-specific T cell hybridomas, which recognize dominant/arthritogenic T cell epitopes. Among these immortalized T cells, hybridoma 5/4E8 (specific for the consensus sequence of 73 GRVRVNSAY in human cartilage PG) induced arthritis upon adoptive transfer, which showed high similarities to the histopathology of the primary form of PG-induced arthritis (PGIA) and those described in peripheral joints of patients with rheumatoid arthritis (RA).To better understand the role of antigen-specific T cells in the development of this autoimmune model of arthritis, we have inserted the Va1.1 and Vh4 chains of the T cell receptor (TCR) of hybridoma 5/4E8 into an in vivo expression vector. We generated transgenic (Tg) mice constitutively (over)expressing both TCR chains. TCR-5/4E8-Tg mice were then backcrossed to the arthritis susceptible BALB/c strain and immunized with cartilage PG. Interestingly, a rapid onset of arthritis with severe clinical symptoms was detected in TCR-5/ 4E8-Tg mice after immunization with PG, which has never occurred in wild-type BALB/c mice. The arthritis was characterized by a chronic progressive disease course with intermittent spontaneous exacerbations and remissions reminiscent of the clinical appearance of RA. Histological analysis of inflamed joints showed extensive cartilage and bone erosions similar to that seen in arthritic joints of human patients. Both IL-4 and IFN-g cytokine-producing cells, with the predominance of IL-4-secreting cells, were detected during the prearthritic stage (initiation phase) of arthritis, which then shifted significantly toward a Th1 bias at the time of onset of arthritis. We also demonstrated that adoptive transfer of splenocytes from arthritic or non-arthritic TCR-5/4E8-Tg donor mice to syngeneic BALB/c-SCID or -RAG-2 knockout recipient mice could induce arthritis.In conclusion, the presence of the large number of arthritogenic epitope-specific T cells with high expression level of epitopespecific TCR is sufficient to induce arthritis. These arthritogenic epitope-specific TCR-5/4E8-Tg mice are valuable and powerful tools, and are now used for further development of T cell directed immune modulating strategies. A series of studies suggests that insulin is a key target in the development of anti-islet autoimmunity in type 1 diabetes of mouse and man. The majority of the Wegmann CD4 T cell clones from islets of NOD mice react with insulin and more than 90% of these react specifically with insulin peptide B:9-23. Using the prototypic I-A g7 -restricted T cell receptor (TCR) of the BDC 12-4.1 anti-B:9-23 clone, we have produced separately and combined a (AV13S3 AJ53) and h (BV2S1 Jh2.1) TCR transgenics. Expression of the Va chain was verified by RT-PCR of splenic mRNA and sequencing of the entire a chain. OR Flow cytometry of peripheral blood mononuclear cells demonstrates good expression of the h chain transgene in T cells. Approximately 98% of the peripheral CD4 T cells in tg FVB mice express the transgenic h-chain compared to 4% in nontransgenic mice. The transgenic FVB mice were crossed and back-crossed with NOD RAG1-/-mice. Homozygous RAG-/-TCR+ mice are lymphopenic compared to heterozygous RAG F TCR+ mice (mean lymphocyte count 500 lymphocytes/ul versus 4800 lymphocytes/ul; P = 0.02). Heterozygous RAG F TCR+ transgenic mice show insulitis at every age tested (7-62 weeks) but do not progress to diabetes nor do they develop insulin autoantibodies. TCR+ RAG-/-tg mice can develop diabetes at older ages (e.g., 32 weeks). Since the insulin2 gene (Ins2) is expressed in the thymus (the insulin1 gene is expressed minimally if at all in the thymus), the TCR transgenic mice were bred with NOD Ins2 knockout mice to create a TCR+ RAG-/-Ins2-/-mouse. Diabetes developed much earlier in these mice (10 weeks for first diabetic observed) with partial restoration of peripheral lymphocytes. We believe the accelerated diabetes and higher peripheral lymphocyte counts of the Ins2 knock-out transgenic mice are due to lymphocytes escaping negative thymic selection due to the lack of insulin expressed as an autoantigen in the thymus. We are currently producing BDC 12-4.1 TCR congenics on the NOD and B6.NOD-H2 g7 backgrounds and will analyze the clonality of the T cell receptors of the infiltrating cells in insulitic lesions. These experiments indicate that as a transgenic, the 12-4.1 T cell receptor confers diabetes susceptibility in immune-compromised mice and confers insulitis susceptibility in immunocompetent mice.OR-88. Sarcoidosis is a systemic granulomatous disease with unclear etiology and limited treatment. Th1 cell activity plays prominent role in the multi-organ inflammation of sarcoidosis, but mechanistic study and therapeutic progress is hampered by the lack of an experimental animal model. We recently reported that Gai2-/-T cells produced chronic intestinal inflammation when transferred into RAG2-/-recipients. In this study, we demonstrate that re-transfer of splenic T cells from these recipients induce progressive systematic granulomatous disease. The inflammation involved skin, lungs, pancreas, and intestines. By flow cytometry, lymphocytes recovered from the mice with disease were increased in CD4 + T cells with Th1 features. Surprisingly, co-transfer of wildtype mesenteric node (MLN) B cells prevented CD4 T cell expansion, inflammation, and disease activity induced by the immunopathogenic lymphocytes. The protective function of MLN B cells required genetic sufficiency for CD1d and IL-10. These results establish a mouse model for sarcoidosis, and reveal a new setting for protective B cell immunoregulation via cognate CD1d interaction and IL-10 production. This model provides an experimental system to delineate immune targeting and immunoregulatory deficits that may underlay pathogenesis in sarcoidosis. Supported by NIH DK46763, DK069434, and the CrohnTs and Colitis Foundation of America. The one yearincidence (10-30%) of this radiation-induced AML (RI-AML) markedly increased (50 % and 75%), when irradiation was followed by treatment with corticosteroids or CSF-1 [1, 2] . A secondary proliferative stimulus then results in leukemic transformation [3, 4] .Aim: This study was initiated to investigate whether allogeneic bone marrow transplantation (allo BMT) can be used as a proliferative stimulus after irradiation of SJL/J mice, thereby creating a model of endogenous post transplant leukemia relapse.Materials and Methods: SJL/J mice (H-2K s ) were sublethally irradiated (8, 5 Gy) and transplanted with 10 7 T-cell depleted Balb/c BM cells (H-2K d ). Animals were monitored for weight loss, signs of leukemic disease and survival. At regular time intervals, peripheral blood was collected for evaluation of donor chimerism in different cell lineages (flowcytometry). Moribund animals were sacrificed and lymphoid and other tissues were prelevated for flowcytometric (CD3, CD4, CD8, CD11b, Gr1, c-Kit, Vb 8.3, H-2K d , IA d ), histopathological and immunohistochemical studies (HE stains; MPO, B220 and CD3).Results: BMT led to the development of mixed chimerism of 10 F 4.5, 80 F 17.3 and 97 F 1.5 % in the T-cell, B-cell and myeloid cell lineage, respectively. From 3 weeks after BMT onwards, all mice developed weight loss and overt malaise (n = 65), resulting in 60% mortality between day 30 and 60 post BMT. Autopsy of these mice showed an enlarged spleen with nodules and a brownish discoloration of the liver with necrosis. Microscopic studies showed destruction of the splenic architecture by a uniform cell population, which was also found in the liver. The hosttype origin and the immature myeloid nature of this population was demonstrated using immunohistochemistry (MPO +, B220-, CD3 -) and flowcytometry (CD11b lo, Gr1 lo, B220 -, CD3 -, CD4 -, CD8-and H-2K d -) on spleen, liver and bone marrow. Ex vivo MLR failed to reveal increased alloreactivity and in vivo expansion of alloreactive T cell frequency (Vb8.3) was absent, arguing against graft-versus-host disease.Conclusion: We describe a new model of endogeneous leukemia, in which irradiation and allo BMT in SJL/J mice gives rise to fatal AML. The model most likely involves a radiationinduced defect, while endogenous growth factors, produced after allo BMT, play a facilitating role in leukemogenesis. This model can be of value for the study of leukemogenesis and of immunotherapy in AML. T and B cells that negatively regulates their function. TIRC7 is upregulated in vivo in patients with Rheumatoid Arthritis (RA) and mice with collagen induced arthritis (CIA) suggesting TIRC7 targeting might be a new therapeutic option of RA. Methods: We analysed the in vitro, ex vivo, and in vivo effects of anti-TIRC7 mAb particularly on memory T cell function under physiological and pathophysiological (CIA model) conditions. Results: Antibody targeting of TIRC7 in vitro significantly inhibited the memory T cell response to recall antigens in vitro and inhibited a delayed type hypersensitivity response in vivo (mouse). Most importantly, DAB mice with established collagen-induced arthritis (CIA) were treated with TIRC7 mAb alone or in combination with a TNF-alpha receptor-Ig-fusion protein.Anti-TIRC7 antibody administration demonstrated significant therapeutic efficacy in established CIA as monotherapy. Moreover, the combination of anti-TIRC7 antibody with a TNF alpha receptor-fusion protein revealed additional therapeutic effects in established arthritis in mice. Mice treated with anti-TIRC7 mAb also showed a significant reduction of IgG1 anti-collagen antibody responses together with reduced B cell numbers. Conclusion: The treatment of autoimmune diseases such as RA associated with exaggerated T and B cell response with an anti-TIRC7 mAb might be unique as TIRC7 targeting results in modulation of both T and B cell response. Moreover, unlike to other therapeutic pathways, anti-TIRC7 antibody therapy exhibits a significant inhibitory effect on memory T cell activation. TIRC7 targeting could offer a novel therapeutic strategy for RA patients that synergizes with TNF alpha receptor therapy and the combination of TIRC7 signaling pathway with TNF alpha blockade might be important for the clinical use as a large group of non-responders to anti-TNF alpha targeting therapy is existing. PD-L1, also known as B7-H1, is one of the ligands of programmed death 1 which can negatively regulate lymphocyte activation. PD-L1 is broadly expressed in mice and may contribute to the peripheral tolerance by interacting with PD-1. Based on this PD-1-mediating immune-inhibitory function, we investigate the preventive and/or therapeutic potential of PD-L1 in autoimmune diabetes. In anti-CD3 stimulation experiment, proliferative response of splenocytes from non-obese diabetic (NOD) mice is down-regulated in a PD-L1.Ig dose-dependent manner. We further generated the transgenic NOD mice overexpressing PD-L1 in pancreatic cells and characterized the protective potential in these mice. In these transgenic mice, we observed an islet-specific transgene expression of PD-L1 both in transcriptional and translational levels. Strikingly, the severity of insulitis in these transgenic mice is significantly decreased. Moreover, the disease onset is delayed as well as the diabetic incidence is decreased in these mice. To assay whether the protection of diabetes in these mice is differentially regulated by the Th1 and Th2 development, we crossed PD-L1 transgenic mice with T1 and T2 doubly transgenic NOD mice and directly investigated their Th1 and Th2 expression profiles. In the PD-L1/T1/T2 triply transgenic mice, the higher Th2 marker expression suggests that overexpressed PD-L1 may indirectly trigger the Th2 development. By enhancing the Th2 function, the original Th1-dominant autoimmune response can be suppressed in these PD-L1 transgenic mice. Furthermore, the transgenic islets had a higher transplantation success rate and survived for longer than wild-type islets. Our results support the theoretical basis for genetic manipulation in an organ-specific manner and provide a potential therapeutic approach mediated by PD-L1 in islet transplantation. PD-1, a member of the B7/CD28 family of costimulatory molecules, is expressed on activated T and B cells and plays a role in regulating tolerance and autoimmunity. PD-L1 is widely expressed on both immune and non-immune cells. PD-L2 has more restricted expression and is primarily found on macrophages and dendritic cells. PD-L1 is upregulated on islet cells in diabetic NOD mice. To investigate the role of PD-L1 and PD-L2 in progression to autoimmune diabetes, we crossed PD-L1/PD-L2 deficient mice onto the NOD background. Both wild type and PD-L1/PD-L2 deficient T cells are diabetogenic when adoptively transferred into PD-L1/PD-L2 deficient NOD SCID hosts, while wild type and PD-L1/PD-L2 deficient T cells do not induce diabetes when adoptively transferred into wild type NOD SCID hosts. These data indicates that PD-L1/PD-L2 expression is required on host tissues (islet cells, endothelium, dendritic cells) in order to dampen autoreactive T cell responses. Respiratory viruses such as influenza A elicit an immune response involving the activation, proliferation and recruitment of CD4 and CD8 T cells to the lung, the major site of viral infection. A major feature of the anti-viral response is the secretion of IFN-g, the signature Th1 cytokine, by activated effector CD4 and CD8 T cells. We have previously observed that these T cells can persist and spontaneously secrete IFN-g in the lungs for up to 30 days following viral infection, and have reported that IFN-g induced by viral infection contributes to major quantitative and qualitative alterations of pulmonary dendritic cells (Nat. In the current study, we have determined how the inducible costimulatory molecule,(ICOS) regulates CD4 and CD8 T cell responses following influenza A infection. Substantial levels of ICOS were observed on CD4 and CD8 T cells during the acute influenza viral infection, which persisted for at least 30 days postinfection, subsiding gradually in a manner that paralleled the expression of IFN-g. We neutralized ICOS-ICOS ligand interactions during acute influenza viral infection of BALB/c mice using either recombinant ICOS-Ig fusion protein or neutralizing anti-ICOS mAbs. Although there was initially a delayed recruitment of CD4 T cells, this was followed by substantially increased number of CD4 T cells in the lungs. Moreover, intracellular staining of these T cells demonstrated an elevated level of spontaneous production for both IFN-g and the immunoregulatory cytokine IL-10 for up to 30 days post-influenza infection. Thus, transient ICOS blockade dramatically alters the normal temporal expression of IFN-g by CD4 and CD8 T cells. To further investigate the effects of ICOS-ICOS ligand interactions in vitro, we activated ovalbumin (OVA)-specific DO11.10 CD4 T cells by co-culturing them with antigen and dendritic cells in the presence of blockade of ICOS/ICOS-ligand interactions. This blockade enhanced antigen-specific proliferation of the CD4 T cells and secretion of IFN-g. The in vitro primed CD4 T cells that received ICOS blockade were adoptively transferred in vivo and recipient mice were then sensitized and challenged with intranasal injections of OVA. CD4 T cells were purified from the pulmonary lavage of the mice and the adoptively transferred cells were found to express higher levels of IFN-g and IL-10 compared to adoptive transferred CD4 T cells that were not subjected to ICOS/ICOS-ligand blockade. Together, these results suggest a novel and previously unappreciated role for ICOS in negatively regulating both Th1 and regulatory cytokine production by T cells. The newly identified Tim (T cell, immunoglobulin and mucin domain-containing molecule) gene family has been associated with the regulation of TH1 and TH2 immune responses. Tim-1 has been genetically linked to asthma, a TH2-mediated disease; however, its endogenous ligand has not been identified. We have found that Tim-4, which is expressed by antigen-presenting cells, is the ligand for Tim-1. An interaction between Tim-1 and Tim-4 can be observed between in vivo-derived cells and can be specifically blocked by anti-Tim-1 antibody. In vivo admin-istration of soluble Tim-1 fusion protein (Tim-1Ig) during either a TH1-or TH2-biased immune response results in hyperproliferation and the preferential expansion of TH2 cells. Furthermore, soluble Tim-4Ig can costimulate T cell expansion both in vitro and in vivo. Our data suggest that the Tim-4/Tim-1 interaction delivers a signal necessary for the expansion of T cells. Aim: Mixed chimerism and donor-specific tolerance can be achieved by bone marrow transplantation (BMT) with nonmyeloablative conditioning using 3 Gy total body irradiation (TBI) and anti-CD154 mAb. CD4 T cells are needed during the first 2 weeks for CD8 T cell tolerance induction. We now investigated early CTL activity and the role of various donor cell populations in this tolerance model.Methods: Recipient B6 mice were treated with TBI (3 Gy, day -1), one dose of anti-CD154 mAb (2mg MR1, day 0) and BMT from a fully allogeneic (B10.A) donor to induce mixed lymphohematopoietic chimerism. CTL function was assessed by 51 Cr release assay on days 4, 7 and 14 after BMT. To assess the role of specific donor cell populations in tolerance induction, recipient B10.S mice received the same conditioning followed by BMT from fully allogeneic B cell-or T cell-deficient mice (A MT or TCRh-/-, B6 background). In a further experiment, transgenic mice expressing the diphtheria toxin receptor under a CD11c promotor were used as donors. In these mice diphtheria toxin injection leads to rapid depletion of CD11c + dendritic cells.Results: In earlier studies we showed that donor-specific CD8 T cells are deleted from the peripheral repertoire within 10-14 days after BMT with this regimen. We now demonstrate donor-specific loss of CTL function in 51 Cr release assay already by days 4 and 7 after BMT in mice that received the tolerance protocol, but not in those receiving conditioning without BMT. This finding suggests an early phase of donor-reactive CD8 T cell anergy before deletion. In search of a tolerogenic donor cell population, we used either B cell-or T cell-deficient donor mice and also tested dendritic cell depletion in a transgenic model system. The absence of any of these cell populations from the donor marrow did not interfere with the establishment of lymphohematopoietic chimerism. Tolerance was further shown by durable mixed chimerism and acceptance of donor skin, but prompt rejection of third party skin.Conclusion: Donor-specific alloreactive CD8 T cells are unresponsive within 4 days and deleted from the periphery within 10 days after BMT with TBI day -1 and anti-CD154 mAb treatment. Neither B cells, T cells nor dendritic cells of donor origin are critically required for tolerance induction in this model. Effective immunity against tumors often results from the development of a vigorous cell-mediated immune response. Effective anti-tumor immunity is largely predicated on the contribution of Th1 cytokines (i.e., IFN-g, TNF-a). While differentiation of T cells toward a Th1 or Th2 phenotype involves a complex chain of events, members of a novel class of molecules called TIMs (T cell Immunoglobulin and Mucin domain-containing proteins) have recently been shown to exert great influence over Th1/Th2 immune phenotype balance in vivo. Manipulating the action of TIM-3, one of the members of this novel protein family, was previously found to profoundly affect disease severity and outcome in animal models of autoimmunity and allograft transplantation, and promote hyperproliferation of antigen-activated T cells and the spontaneous production of Th1 cytokines. These results strongly suggested that agents targeting TIM-3 pathways could also enhance effective anti-tumor immune responses in vivo, presumably through one or more mechanisms favoring Th1 cells and the promotion of cell-mediated immunity. In the study described here, an antibody specific to TIM-3 was investigated for its ability to promote anti-tumor effects in mice. Using the EL4 thymoma tumor model, anti-TIM-3 was delivered as either a standalone therapeutic agent following establishment of tumors under the skin, or as an adjuvant to irradiated tumor cell vaccination prior to live tumor challenge. Anti-TIM-3 antibody promoted significant reductions in challenge tumor growth over time. Furthermore, including anti-TIM-3 as an adjuvant to tumor vaccination also allowed treated mice to fully reject subsequent live tumor challenge. Neither tumor rejection nor limited tumor growth was seen in mice receiving isotype-matched control antibody under either experimental protocol. By demonstrating a powerful capacity for TIM-3specific antibodies to change the course of tumor progression in treated mice, these experiments further support the prospective role of TIM-3 to act as a critical regulator of cell-mediated immune function and Th1 responsiveness. MS is an inflammatory disease of the CNS white matter, with presumed autoimmune etiology. Therapies in current use show benefit for MS in targeting the T-cell autoimmune response. Apoptosis of auto-reactive T-cells is a fundamental immunoregulatory mechanism. If autoimmune T-cells were predisposed to death, MS could be alleviated. Increased expression of Inhibitor of Apoptosis (IAP) proteins protects cells from apoptosis. In particular, the X-linked IAP (XIAP) interrupts the apoptotic cascade in T-cells by directly inhibiting effector caspases. Inhibition of XIAP primes cells for apoptosis induced by multiple stimuli. We used repeated interperitoneal (IP) injection of XIAP antisense oligonucleotide (AEG35169) in mice to reduce XIAP protein levels in peripheral blood leukocytes. AEG35169 was similarly administered to treat MOG p35-55 induced EAE in C57Bl/6 mice. When given daily via IP injection, from time of symptomatic onset, AEG35169 reduced clinical scores within 5 days and prevented further disease progression in 84% of animals, compared to control groups receiving random or scrambled oligonucleotides or saline, 90% of which showed continued disease or increased severity (n = 16). Anti-XIAP treated animals showed evidence of considerably increased leukocyte apoptosis, with high numbers of TUNEL positive cells in the spinal cord. A 5day prophylactic treatment with AEG35169, prior to induction of EAE, followed by daily treatment, reduced the incidence of mild disease from 85% of animals to 9% (n = 57), and of severe disease from 84% to 38% (n = 48). Analysis of tissues at 40 days after immunization indicated no or very limited inflammatory infiltrates in anti-XIAP protected animals, and correlates of disease such as chemokine expression in CNS were reduced in treated animals. Amelioration of disease was not due to immune suppression, and there was no evidence for a Th1/Th2 shift in treated animals. Our data establish XIAP as a critical controller of the susceptibility of CNS-infiltrating T cells to apoptosis, such that experimental modulation of XIAP prevents or cures EAE. These studies increase our understanding of regulation of inflammatory pathology in the CNS and support XIAP as a novel target for therapeutic intervention in MS.OR-101. Anti-IL-2R Therapy: An Alternative Strategy for Regulating CD40L Expression.J. Shen, 1 J. T. Snyder, 1 H. Azmi, 1 J. 1 1 Laboratory of Immunology, NEI, National Institutes of Health, Bethesda, MD, USA.Monoclonal antibodies directed against the alpha chain (Tac/ CD25) of the IL-2 receptor (IL-2R) are an emerging therapy in both transplantation and autoimmune disease. In a cohort of patients with autoimmune uveitis being treated with multiple immunosuppressive medications, monotherapy with daclizumab, a humanized anti-Tac antibody, was sufficient to control their disease without serious side effects. The basis of this antibodyTs therapeutic efficacy has not been established. Meanwhile, antibodies against CD40L that were shown to be efficacious in primate transplant models were withdrawn from clinical use due to serious side effects associated with their administration. We have reported that CD40L expression on activated human CD4+ T cells is biphasic, consisting of an early CD28-independent peak, a subsequent nadir, and a second, CD28-dependent peak at 48 hr. The transient expression of CD40L is critical to the physiologic function of this costimulatory pathway, yet the mechanisms underlying the biphasic pattern of CD40L expression are largely unknown. We have also reported that the CD28-dependent second phase of CD40L expression is severely inhibited in vitro by daclizumab. We now show in primary PBMC cultures using blocking antibodies and flow cytometry that IL-2 does not impact late phase CD40L by acting indirectly through IL-2 regulated Th1 and/or Th2 cytokines as neither IL-12 nor IL-4 had any appreciable effect on either early or late expression of CD40L. In addition, CD28 signaling is not necessary for late phase CD40L expression as recombinant IL-2 or an agonistic anti-CD40 mAb could substitute for CD28 costimulation. Finally, in contrast to earlier reports, we observe that down regulation of early CD40L expression is not dependent upon interactions with CD40. These results suggest that daclizumab, in combination with agents that can block early CD40L expression, may be a viable alternative to the use of anti-CD40L antibodies clinically. Background and Objectives: Rheumatoid arthritis (RA) is an autoreactive disease in which activated T cells play an important role orchestrating the autoimmune responses giving rise to the inflammatory cascade responsible for joint inflammation and bone destruction. The CD28/B7 costimulatory pathway is critical for full T cell activation and modulating this pathway has been shown to inhibit T-cell activation leading to inhibition of these immune responses. Abatacept modulates T cell activation by interfering with the engagement of CD80/86 with CD28. Abatacept has been shown to provide significant improvement in the signs and symptoms of rheumatoid arthritis in a phase II trial. Here, we examine the effect abatacept administration has on disease induction, anti-collagen antibody production and bone destruction in a rat model of collagen induced arthritis.Methods: Female DA rats were immunized s.c. on day 0 with 300 ug of bovine type II collagen in incomplete FreundTs adjuvant at the base of the tail. Immunized rats were administered either 1 mg/kg abatacept or a control human IgG IP on days -1, 0, 2, 4, 6, 8 and 10 . with a plethysmometer. Both hind paws were measured and the change in volume from base-line measurements (Day 0) were recorded. At the conclusion of the study (day 27) serum samples were collected from each animal for measurement of collagen specific antibodies by ELISA as well as serum cytokine measurements. Legs from the rats were removed and placed in formalin and prepared for histological analysis as well as analysis of bone morphology by micro CT.Summary: By day 16 of the study, significant paw swelling was observed in the IgG treated control animals and continued to increase throughout the study until reaching a plateau (~3-3.5 mls.) The IgG treated rats reached 100% incidence while no incidence was observed in the abatacept treated group. Serum anti-collagen antibody levels correlated well with the paw swelling data where abatacept administration resulted in 90% inhibition of collagen specific antibodies. We also found that abatacept decreased the expression of many of the circulating cytokines and chemokines which were upregulated in diseased animals. In these studies, paw swelling, collagen specific antibodies and bone destruction were all inhibited by the treatment.OR-103. Abatacept (CTLA4Ig) Modulates Human T-Cell Proliferation and Cytokine Production but Does Not Affect TNFA Production by Monocytes. P. M. Davis, 1 S. G. Nadler, 1 K. A. Rouleau, 1 S. J. Suchard. 1 1 Immunology and Inflammation Drug Discovery, Bristol-Myers Squibb, Princeton, NJ, USA.Background and Objectives: Activated T cells play a central role in the inflammatory cascade leading to joint inflammation and destruction characteristic of rheumatoid arthritis (RA). The cytokines secreted by activated T cells can both initiate and propagate the immunologically driven inflammation associated with RA.Abatacept, the first of a new class of agents that selectively modulate the co-stimulatory signal required for full T-cell activation, was evaluated in vitro for its ability to regulate human T-cell proliferation and cytokine production. The effect of abatacept on immune complex (IC)-or LPS-induced TNFa release from monocytes was also evaluated to distinguish the impact of abatacept on innate versus adaptive, antigen-specific responses.Methods: T cells were isolated from normal healthy volunteers. The effect of abatacept on antigen-dependent T-cell activation was evaluated using either an irradiated human B-cell line (PM-LCL) as the antigen-presenting cells (APCs) for a primary mixed lymphocyte reaction (MLR), or autologous E-PBMCs as APCs, for a recall response to tetanus toxin (TT). Cytokines were measured at various times post activation, with proliferation determined on day 5. Monocytes were isolated by elutriation, challenged with LPS or ICs, and TNFa levels measured at 6 h. Chi L6 was included as a nonspecific Ig fusion protein control.Summary: Abatacept significantly down modulated T-cell proliferation, in both primary and recall responses, at concentrations between 0.3 and100 Ag/ml, with maximal inhibition (~60-80%) observed at~3-10 Ag/ml. These concentrations are below the abatacept trough plasma levels observed in patients receiving a clinically effective dose. 1 Under conditions of maximal inhibition of proliferation, and similar to trough plasma levels in patients (30 Ag/ml), abatacept inhibited cytokine production in both primary and TT-dependent recall responses. However, the extent and rank order of cytokine inhibition by abatacept was markedly different between these two responses. Specifically, inhibition of IL-2 N TNFa N IFNg in a primary response whereas inhibition of IFNg z IL-2, with a minimal effect on TNFa production in a TT recall response. In contrast, abatacept did not inhibit IC-or LPS-induced TNFa production in human monocytes.Conclusion: Abatacept, a selective co-stimulation modulator significantly inhibited the activation (as measured by cytokine production) and proliferation of human T cells in the context of a primary MLR or TT-dependent memory response. This inhibition occurred at concentrations below the serum C min levels observed in patients receiving a clinically effective dose of abatacept 1 (10 mg/kg monthly), consistent with suppression of T-cell activation in vivo. There was no effect of abatacept on TNFa production in monocytes challenged with LPS or ICs indicating that this agent may largely preserve innate immune responses. Kremer JM, et al. Essential Role of IL-10 in Restricting Immunity during a Chronic Viral Infection.Peripheral blood mononuclear cells were isolated and stimulated with streptococcal antigens, superantigens or the mitogen PHA in the presence of 0-10 mM MTX. T cell expression of adhesion molecules and activation markers, and the amount of T cell apoptosis in cultures, were determined flow cytometrically. The folate-and adenosine-dependent pathways of MTX action were manipulated using specific agonists and antagonists.We show that MTX caused a dose-dependent suppression of T cell activation and adhesion molecule expression, and this was not due to T cell apoptosis. The suppression of intercellular adhesion molecule (ICAM)-1 was adenosine and folate-dependent, while MTX suppression of the skin-homing cutaneous lymphocyteassociated antigen (CLA) was adenosine-independent. The effect of MTX on CLA, but not ICAM-1, required the constant presence of MTX in cultures.The suppression of T cell activation and T cell adhesion molecule expression, rather than apoptosis, mediated in part by adenosine or polyglutamated MTX, or both, are important mechanisms in the anti-inflammatory action of MTX. Integrins are heterodimeric transmembrane proteins that regulate cell-cell and cell-matrix interactions. The alpha (v) containing integrins represent a major family of RGD-binding integrins, and have been shown to have important roles in angiogenesis, tumorigenesis, neural development and wound healing. Alpha (v) beta (3) is expressed by many immune cells and surface expression is highest in tissue resident cells such as gy T cells and B1 B cells. Various alpha (v) integrins are expressed by monocytes, macrophages and DCs, and have been shown by antibody blockade to regulate monocyte transmigration and phagocytosis of apoptotic cells by macrophages and DCs. However definitive in vivo studies of the role of alpha (v) in the immune system have been limited by the lethality of alpha (v) knockout mice. Here we describe the generation of conditional knockout mice to study the role of these adhesion molecules as regulators of leukocyte function. Conditional deletion of alpha (v) using mice expressing CRE from the Tie2 promoter generated mice lacking alpha (v) in endothelial cells and all hematopoietic cells. Although these mice appeared normal at birth, they developed signs of chronic disease and weight loss beginning at 8 weeks, which progressed such that 75% of experimental animals had died by 40 weeks. Pathological examinations revealed that the mice had developed spontaneous transmural gastro-intestinal (GI) and respiratory tract inflammation, ulceration and epithelial cell hyperplasia. These histological findings, in combination with the clinical observations of wasting and GI obstruction are consistent with chronic progressive inflammatory bowel disease (IBD). To further define this phenotype we have selectively deleted alpha (v) in specific leukocyte compartments and our results suggest that alpha (v) integrins regulate both cell migration and cell responses to pathogen derived ligands. In conclusion these data demonstrate that alpha (v) plays an essential role in regulating immune homeostasis in the GI and respiratory tracts, and that deletion of alpha(v) generates a new model of spontaneous, chronic IBD. We therefore propose a novel function for alpha (v) integrins in the normal regulation of inflammatory responses and immune homeostasis. Food allergy is a significant health problem, particularly in Western countries. In the UK and the USA, peanuts are a common cause of food allergy, associated usually with high titer IgE antibody and consistent with the preferential activation of T helper (Th) 2 type cells. Using high IgE responder BALB/c strain mice, we have previously shown that sensitization with peanut lectin (a minor peanut allergen) is associated with an increase in allergenspecific IgE and changes in cytokine protein and mRNA expression indicative of a selective Th2 type response, with elevated levels of IL-4, but not IFN-g, cytokine expression. Here we have used flow cytometric analyses of intracellular cytokine expression patterns to determine the relative contributions of CD4 and CD8 T lymphocytes to the immune phenotype that develops following exposure to peanut lectin.METHODS. Healthy PBMCs were incubated with the serum of SOJIA patients. Changes in gene transcription were assessed using oligonucleotide microarrays and real-time PCR. Genes whose expression was most significantly altered were identified and analyzed in the PBMCs of 16 SOJIA patients and 12 healthy controls. Additionally, we have performed global gene expression analysis using blood PBMC RNA from 31 SOJIA patients to hybridize Affymetrix U133 gene arrays. One third of the patients were in remission, one third had systemic symptoms and the remaining one third had polyarticular arthritis with no systemic involvement at the time of analysis. We also compared the gene expression of these patients to children with systemic infections. PBMCs from healthy controls and SOJIA patients were exposed in vitro to PMA/Ionomycin to assess their cytokine secretion capacity. Twelve SOJIA patients were treated with Anakinra for 2-16 months.RESULTS. SOJIA serum increased the transcription of IL-1a, IL-1b and other innate immunity genes. Several of these genes were upregulated in vivo in the PBMCs of SOJIA patients. Treatment of 12 SOJIA patients during the systemic and/or arthritic phase of the disease with Anakinra for a period of 2-16 months induced complete disease remission in 9/12 patients and a partial response in the remaining 3 patients.CONCLUSION. Human tumours over-express a variety of TAAs (Tumour Associated Antigens), which are either absent or expressed at low levels in normal tissues. Peptides derived from TAAs are presented on the surface of tumour cells by Class I HLA molecules, and represent targets for cytotoxic or immunotherapeutic anti-cancer agents. NY-ESO is a TAA of unknown function, over-expressed in a number of tumour types, including melanoma and bladder. We have generated a soluble TCR (T Cell Receptor) specific for an NY-ESO peptide presented by HLA-A2. The TCR lacks transmembrane domains and is stabilised by a novel disulphide bond; it is expressed in E. coli as separate a and h chains, and refolded from inclusion bodies. The natural affinity of the TCR is 24AM; in order to generate a molecule suitable for targeting tumours, the TCR was affinity matured using phage display technology. We show data to demonstrate that the TCR binds specifically to NY-ESO peptide pulsed cells (by FACS analysis), and also that it targets tumour cells expressing NY-ESO, by fluorescence microscopy. Furthermore, the TCR specifically inhibits activation of a T cell clone by NY-ESO +ve tumour cells, measured by INFg ELISPOT. Immune activators, including cytokines, have been fused to the TCR h chain C terminus; these fusion proteins are suitable for development as immunotherapeutic agents, to treat NY-ESO +ve tumours. In type 1 diabetes the major loss of insulin producing beta cells is caused by autoreactive T-cells specific for antigens expressed by the pancreatic islets. In this study we have analyzed the prevalence of GAD65-and proinsulin-specific CD4+ T-cells in type 1 diabetic patients, prediabetic subjects (positive for two or more autoantibodies) and in HLA-genotype matched islet-cell autoantibody (ICA) negative healthy children. Peripheral blood mononuclear cells, from DRB1*0401, 0404 or 0301 positive children in these three study groups, were cultured in the presence of two different GAD65 peptides (557I; aa 555-567 and aa 274-286) or with a proinsulin (aa 24-36) peptide for 10-11 days. Thereafter the cells were restimulated with MHC class II monomers for 3 days. The monomers contained the same peptides as used in the primary stimulation. Binding of CD4+ T-cells to GAD65 or proinsulincontaining MHC class II tetramers was analyzed by flow cytometry. Our results show that 11 of 18 (61%) type 1 diabetic patients and 7 of the 20 (35%) prediabetic subjects were positive for one of the GAD65 or proinsulin-containing tetramers, whereas only 3 of 23 (13%) ICA negative healthy controls had tetramer binding cells. The difference between type 1 diabetic patients and healthy controls was statistically significant (P = 0.004, Chisquare test). The frequency of tetramer positive cells in the GAD65 or proinsulin activated CD4high/CD25+ cells was higher in type 1 diabetic patients (0.00-9.19% ) and in prediabetic subjects (0.00-53.60%) than in control subjects (0.00-2.84%) (P = 0.01 and P = 0.03 for respectively study group, Mann-Whitney U-test). In conclusion, type 1 diabetic patients and prediabetic subjects have a higher prevalence of GAD65-and proinsulin-specific CD4+ T-cells than HLA-genotype matched healthy controls. skin prick test (SPT) with a series of common allergenic extracts including grasses, weeds, trees, house dust mites and moulds. Results: 132 subjects (62.2%) had positive SPT to at least one aeroallergen. The prevalence rates for allergen groups were: pollens (92.4%), mites (22.7%) and moulds (8.3% Mast cells play pivotal roles in immediate-type and inflammatory allergic reactions that can result in asthma. Cross-linking of the high-affinity receptor for IgE (FcRI) on mast cells activates a signaling pathway leading to Ca 2+ mobilization and is followed by degranulation and the release of histamine and other preformed mediators, as well as de novo synthesis of the arachidonic acid metabolites i.e leukotrienes, and prostaglandins. To investigate possible effects of heat shock on immunologic functions of bone marorow derived mast cells (BMMC), we studied degranulation and leukotrien production. Blockade of TNFA Preferentially Inhibits Proliferation of Anti-CD3, Recall-Antigen Responsive and Autoreactive Human VLA-1+CD45RO+CD4+ T Cells.S. 1 1 Medicine, Chaim Sheba Medical Center and Tel Aviv University, Ramat Gan, Israel; 2 Medicine, Columbia University, New York, NY, USA.The very late antigen (VLA)-1 a1h1 integrin, a receptor for collagen, is induced on the surface membrane of activated T-cells (TC) and remains preferentially expressed by effector-memory Th1 cells. We recently showed that VLA-1+ T cells at sites of chronic autoimmune inflammatory arthritis express a restricted and unique T cell receptor (TCR) Vh repertoire, suggesting they are responding to a unique set of auto-antigens in tissues. Since VLA-1+ immunocytes are critical in immune mediated Th1 diseases that are ameliorated by monoclonal antibodies (mAb) to TNFa, we explored how an anti TNFa mAb affects the VLA-1+CD4+ TC subset. Anti TNFa mAb (Infliximab, 5-50 Ag/ml) but not control immunoglobulins, neutralized TNFa during ex vivo mitogen (PHA or anti-CD3)-triggered activation of VLA-1-peripheral blood (PB) mononuclear cells (MC) (PBMC) and significantly reduced the percentage of VLA-1+ TC in 8-12 day cultures (36.9 F -20.3% to 26.9 F 15.7% of the TC, n = 9, pb0.011), but did not affect the VLA-4+ subset. Furthermore, CFSE-dye intracellular labeling revealed that the reduction was due to a preferential inhibition of VLA-1 expression among CD4+ TC that were induced to divide in the presence of anti CD3. Thus, dividing VLA-1+CD4+ T cells in the culture, were reduced 66 F 22% while non-dividing VLA-1+CD4+ TC were slightly increased. In contrast, the anti CD3 VLA-1-CD4+ responsive subset was inhibited to a lesser extent by TNFa blockade (40-50% inhibition, n = 5). The addition, at a 1:2 cell:cell ratio, of washed MC from 8 day cultures of PBMC activated by anti CD3 mAb plus anti TNFa, but not, as a control, of VLA-1-anti CD3 triggered T cells in the absence of anti TNFa, likewise decreased the VLA-1+ subset emerging in autologous de novo anti-CD3-activated 8 day cultures, suggesting that the preferential inhibition of VLA-1+ CD4+ TC division by anti TNFa, may involve MC activated in the presence of anti TNFa (n = 3 experiments). Importantly, anti TNFa mAb, also preferentially inhibited PB derived VLA-1+CD4+ TC dividing ex vivo in response to the recall antigen tetanus toxoid, while less potently inhibiting the VLA-1-CD4+ subset. Finally, non-antigenically or mitogenically stimulated, spontaneously dividing, (thus presumably autoreactive), synovial fluid (SF) VLA-1+CD4+, but neither VLA-4+CD4+or CD25+CD4+ TC, from patients with autoimmune arthritis, were also dramatically and preferentially reduced by anti TNFa (85% inhibition, n = 6) in ex vivo cultures. These data suggest that a critical immuno-modulatory effect of anti TNFa is mediated by its ability to preferentially target and inhibit mitogen, antigen or auto-antigen induced expansion of the VLA-1+ Th1 effector memory subset. Antibodies endowed with specific recognition properties for HLA-displayed tumor associated antigens have been recently produced and shown to directly detect expression of HLA-tumor associated antigens on the surface of cancer cells. The application of these reagents for validation of T cell epitopes would greatly facilitate vaccine development. Yet many technical obstacles stand in the way of developing a consistent approach for making antibodies with T cell receptor (TCR)-like specificity. Particularly important would be to develop immunogenic forms of immunogen that could then be used for rapid and reproducible immunization of mice for the generation of polyclonal antibody responses reactive against peptide-A2 epitopes. We hypothesized that HLA-A2peptide immunogen presented to the immune system in a tetravalent form rather than a monovalent form would display enhanced immunogenicity and promote consistent generation of high titer IgG polyclonal antibody responses specific for the A2peptide immunogen. To test our hypothesis we refolded E. coli produced insoluble protein and prepared purified forms of monomer and tetramer HLA-A2 peptide complexes displaying either human eukaryotic transcription initiation factor 4-gamma (eIF4G; VLMTEDIKL), a self-protein found to be upregulated in HIV infected T cells or human tumor suppressor protein p53 (264; LLGRNSFEV), a self-protein that is widely expressed in many cancers. We then immunized groups of Balb/c mice (5/group) 3 times with 2-week intervals with either monomer or tetramer forms of immunogen and assayed mouse sera for polyclonal IgG antibody response reactive for HLA-peptide by competitive ELISA. All mice (5/5 from both eIF4G-and 264-peptide-A2 groups) immunized with tetramers of immunogen showed specific anti-A2-peptide IgG antibody responses. In contrast, no specific polyclonal antibody response was detected from any of the mice (0/5) immunized with monomers of eIF4G-and 264-peptide-A2 immunogen. To confirm the specificity of the polyclonal antipeptide-A2 response, we evaluated antibody from mice immunized with tetramer immunogen to stain T2 cells (HLA-A0201 positive) pulsed with either eIF4G-or 264-peptide in a competitive binding assay. Our T2 cell assay results support the ELISA data and indicate that immunogen formulated as a tetramer is immunogenic and able to generate anti-peptide-A2 specific antibody responses in mice. Furthermore, we demonstrated by ELISA and T2 cell staining that tetramer forms of immunogen efficiently elicit IgG polyclonal antibody responses reactive against peptide-A2 within 4 weeks after initial immunization. Collectively, our findings support the hypothesis that tetravalent forms of peptide-A2 immunogen consistently lead to specific antibody responses against peptide-A2 epitopes. In addition, the ability to generate these probes in a rapid and reproducible manner will be invaluable for HLA class I epitope validation. Etanercept, a recombinant human TNF receptor fusion protein, is FDA approved for psoriasis and psoriatic arthritis. TNFa increases the synthesis of proinflammatory cytokines and leads to the activation of multiple signaling pathways, including NF-kB. The Rel/NF-kB transcription factors play a central role in numerous cellular processes, including the stress response and keratinocyte proliferation and differentiation. Utilizing a phosphorylation specific antibody we examined the expression of active nuclear NF-kB/RelA via immunohistochemistry in normal skin, non-lesional psoriatic skin, lesional psoriatic skin and lesional skin from patients treated with etanercept. There was no expression of active nuclear NF-kB in normal epidermis, whereas a basal level of constitutive active phosphorylated NF-kB/RelA was present in uninvolved epidermis from psoriasis patients. There was also significant upregulation of active phosphorylated NF-kB/RelA in epidermis from psoriatic plaques. Serial biopsies from psoriasis patients treated with etanercept at 1 month, 3 months, and 6 months demonstrated a significant down regulation of phosphorylated NF-kB/RelA which correlated with decreases in epidermal thickness, restoration of normal markers of keratinocyte differentiation, and clinical outcomes. Reference: Mette Ejrnaes, Matthias von Herrath. 1 Immune Regulation Lab, La Jolla Institute, San Diego, CA, USA.Viruses use a variety of strategies to suppress the anti-viral immune response leading to persistence in the host. Induction of immune suppression is one of the mechanisms by which viruses escape clearance and establish a persistent infection. The cytokine IL-10 has immunomodulatory properties and can down-regulate cellular immune responses by acting on APCs and T cells. To gain further insight into the role of IL-10 during viral persistence we studied lymphocytic choriomeningitis virus (LCMV) infection in its natural host. The LCMV isolate Clone 13 establishes a prolonged infection in Balb/c mice, which is associated with a less effective antiviral immune response and, in some studies, conditioning of dendritic cells. Results: Here, we report that a significant amount of IL-10 is being generated by CD4+ lymphocytes and some classes of APCs during persistent LCMV infection. Treatment with neutralizing IL-10R antibody on days 0, 7, and 14 post Clone 13 infection resulted in accelerated viral clearance. This was associated with a numeric increase of total spleen cells in comparison to non-treated mice and decreased levels of IL-10 were generated by such splenocytes. Lastly, overall clinical appearance was improved through this intervention as reflected in an increase in bodyweight, healthy shiny coat, and increase in physical activity. Conclusion: Our studies indicate that in persistent viral infections IL-10 plays an essential role in suppressing the anti-viral response and that systemic blockade can improve the clinical outcome. A similar strategy might be beneficial in other chronic infections associated with increased IL-10 levels, for example hepatitis C virus infection. This work was supported by an ADA mentor grant and a program project grant from NIAID for Matthias von Herrath. Effects of Natalizumab (anti-VLA-4 Antibody) on Immune Cell Adhesion and Migration in Patients with MS. Trafficking and Adhesion Objective: (1) To establish biological dproof of conceptT that in vivo anti-VLA-4 treatment of patients with multiple sclerosis (MS) results in decreased functional VLA-4 expression and migratory capacity of immune cells, and (2) Develop a simple in vitro assay that could be applied to monitor therapeutic response.Background: Natalizumab (TysabriR), a humanized monoclonal antibody directed against the adhesion molecule VLA-4, has recently been approved for the treatment of patients with relapsing remitting MS. It is presumed that beneficial effects in MS would be based on binding of Natalizumab to VLA-4 on the surface of circulating immune cells, thereby inhibiting their capacity to migrate into the CNS. To date, the impact of in vivo therapy with Natalizumab on the functional expression of VLA-4 on immune cells of MS patients has not been reported. The development of a simple assay to measure this effect could prove very useful in immune monitoring of patients on this emerging therapy.Design/Methods: Consenting patients participating in the open-label phase of a clinical trial of Natalizumab in relapsing remitting MS, provided venous blood immediately prior to (Preinfusion), and one hour following (Post-infusion), monthly Natalizumab infusions (300mg IV). Levels of VLA-4 surface expression on circulating immune cell subsets were assessed by flow cytometry. The migratory capacity of immune cells was evaluated in an established two-compartment Boyden chamber, known to capture VLA-4 mediated migration of human immune cells.Results: We observed that expression of VLA-4 on circulating immune cells was significantly reduced after in vivo Natalizumab infusions (P = 0.004; n = 12). The effect was observed on all immune cell subsets but was greatest on T cells compared to B cells (P = 0.026) or monocytes (P = 0.032). In the functional assay, migration of post-infusion immune cells was significantly decreased compared to the migration of corresponding pre-infusion cells (P = 0.026). The decrease in observed VLA-4 surface expression correlated well with the decrease in migratory function of the corresponding immune cells, following infusions (r = 0.71; p b 0.05). We confirmed that the migration assay can be carried out on frozen mononuclear cells (PBMC), providing a means for monitoring patientsT responses over time. We plan to present a batched analysis of prospectively collected samples from these patients, which should provide insights into the kinetics and stability of these in vivo effects.Conclusions: Our study provides the first biological dproof of conceptT that in vivo Natalizumab therapy results in diminished VLA-4 functional expression and migratory capacity of circulating immune cells. The ability to reproducibly capture this effect in a relatively simple bioassay, and the validation that the assay can be applied to frozen PBMC, could provide a useful means to monitor patients on this promising therapy. Molecular Imaging of Adhesion Molecules in Experimental Autoimmune Encephalomyelitis (EAE). The infiltration of autoreactive T-cells into the central nervous system (CNS) requires a complex molecular interplay between immune cells and the blood brain barrier (BBB), especially involving vascular cell adhesion molecule (VCAM) 1 and intercellular adhesion molecule (ICAM) 1. By combination of SPAQ with specific gasfilled microparticles (MP) targeted against VCAM and ICAM (VCAM-MP, ICAM-MP), we aimed to monitor the molecular changes at the blood-brain-barrier during the course of actively induced or adoptively transferred (AT) myelin basic protein (MBP)-EAE.Ex vivo imaging of ICAM-1 expression in AT-EAE at the disease maximum proved the high sensitivity, specificity and spatial resolution of the method with the possibility of videodensitometric quantification. These results could be reproduced in vivo with a clear periventricular and cerebellar upregulation of ICAM1 and VCAM1 expression at the maximum of AT-EAE which could be suppressed by pretreatment of rats with corticosteroids (P b 0.008). The imaging results were confirmed by parallel immunohistochemistry. Subsequent application of ICAM-MP after ICAM-MP or VCAM-MP injection did not influence follow-up measurements. Sequential imaging of ICAM-MP in vivo over the course of active and AT MBP-EAE revealed a significant upregulation of ICAM before the respective onset of disease (day 2 for AT-EAE, day 10 for active EAE). At that point of time no signal changes were observed on T2-weighted magnetic resonance images (MRI). Albumin staining for detection of BBB integrity and gadolinium enhanced MRI after sonification did not reveal a disturbance of the BBB thereby proving the safety of the method in vivo.Conclusion: Based on these data, molecular imaging of adhesion molecules with SPAQ is a platform technology for quantification of changes at the BBB in vivo with a sensitivity superior to conventional MRI. Experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell mediated autoimmune disease. IL-16 is a CD4+ cell chemoattractant cytokine. Infiltration of the CNS by CD4+ Th1 cells precedes onset and relapses of experimental autoimmune encephalomyelitis (EAE). We reported that (B6 Â SJL) F1 (H-2 b/s ) mice, with severe relapsing-remitting disease, had extensive infiltration by CD4+ T cells compared to C57BL/6 (B6) (H-2 b ) mice, which developed mild low-relapsing disease in response to myelin oligodendrocyte peptide ). This observation led us to search for mechanisms that specifically regulate trafficking of CD4+ T cells in relapsing H-2 b/s mice. In this report we show that the CD4+ cell chemoattractant cytokine IL-16, has an important role in regulation of relapsing EAE induced by MOG 35-55 in the (B6 Â SJL) F1, (H-2 b/s ) mice. Levels of IL-16 in the CNS correlated well with prominent infiltration by CD4+ T cells and B cells during acute and relapsing disease. Infiltrating CD4+ T cells, and occassionaly CD8+ T cells and B cells contained IL-16 immunoreactivity. Pro-IL-16 (80 kD) and cleaved IL-16 (55 and 17 kD) were found in spinal cord of mice with active disease. During remission IL-16 levels were significantly decreased. In relapsing mice, CNS levels of IL-16 peaked and paralleled with activation of caspase-3 and CD4+ T cell infiltration. We identified CD4+IL-16+active-caspase-3+ T cells withn CNS infiltrates. IL-16 (55kD), which co immuno-precipitated with CD4 coreceptor (CD4R), was the most abundant form of IL-16 found during relapse. Our data suggested that IL-16 was produced by infiltrating CD4+T cells through caspase-3 dependent mechanism. It also indicated functional relationship between IL-16 and CD4R, consistent with CD4R specific chemoattractant properties of this cytokine. Based on these observations, we treated EAE mice with IL-16 neutralizing antibody. Treatment with neutralizing anti-IL-16 antibody successfully reversed paralysis and ameliorated relapsing disease. In treated mice, diminished infiltration by CD4+ T cells, lesser demyelination and more sparing of axons were observed. Taken together, we show an important role for IL-16 in regulation of relapsing EAE. We describe a novel therapeutic approach to specifically impede CD4+ T cell chemoattraction in EAE, based on IL-16 neutralization. Immune responses involve multiple cell-cell interactions. We have used explant and intravital confocal or multiphoton microscopy to collect 4D (XYZ and time) data on the interactions of antigen (Ag)-specific T cells with each other and with Ag-bearing dendritic cells (DC) in an intact lymph node (LN). Some naRve T cells move rapidly in the absence of Ag but show prolonged adherence to Ag-bearing DC, accompanied by immunological synapse formation. Activation and detachment from the antigenbearing DC follows, along with cell division. These data suggest that T cell activation follows from prolonged lymphocyte association with individual antigen-bearing DC rather than summation of signals from brief encounters with such presenting cells. CD4 and CD8 T cells associate with a single DC when both antigens are present and direct CD4-CD8 T cell contact is also seen. Formation of these clusters appears to be non-random in nature. Rapid movement of DC dendrites is readily visualized, as is T-DC contact through these processes, followed by movement of the T cell towards the DC body. Quantitative analysis suggests an impact of self-MHC recognition on the time of naRve T cell-DC interaction. Intravital methods have permitted visualization of DC migration into LN and the egress of lymphocytes from HEV for initial contact with DC. Fluorescent reporter constructs (e.g., EGFP under the control of the IL-2 promoter) are revealing the consequences of T-DC interactions in real time within LN and fluorescent chimeric proteins are being used to track redistribution of key proteins during cell movement and interaction. Differential migratory behavior of lymphocytes and DC in distinct regions of the lymph node has been observed, as has the failure of rapidly moving T and B lymphocytes to cross rather strict borders between the T cell zone and B cell follicle. DC distribution and function are being examined in non-lymphoid tissues such as liver (under steady-state conditions and during inflammation) and kidney. The behavior of subepithelial DC in the small bowel has been visualized under steady-state conditions and following infection with Salmonella, which elicits and active protrusion response from the DC. These studies are contributing to a more accurate picture of the molecular, cellular, spatial, and temporal aspects of cell interaction and signaling events in host immune responses. Regulatory T cells (Tregs) have fundamental functions for suppression of immune responses, however, the compartments at which they exert their suppressive functions in vivo are not known. The integrin a E h 7 unravels a fundamental dichotomy between naive-like and effector/memory-like CD4 + Tregs, where only the latter are capable of suppressing Th1-mediated inflammatory immune responses. Strikingly, suppressive action of a E + Tregs is completely dependent on their inflammation-seeking capacity: Tregs from fucosyltransferase-deficient animals, which lack selectin-ligands and fail to migrate into inflamed sites are unable to mediate suppression. In contrast, naive-like a E -CD25 + Tregs, which show an enhanced recirculation through lymph nodes, are more efficient in preventing priming of naive CD4 + T cells. Low-dose methotrexate (MTX) is an established and highly effective treatment for severe psoriasis and rheumatoid arthritis; however, its mechanism of action remains unclear. When used for the treatment of psoriasis, MTX was thought to act directly against the epidermal hyperproliferation, however, the poor efficacy of locally administered MTX and the effectiveness of agents that target T cells strongly suggest that the anti-proliferative effect of MTX is not responsible for its efficacy in psoriasis. Therefore, we investigated the effects of low-dose MTX on T cells and explored through which cellular pathways these effects are mediated. Cytokine Mediated Immunoregulation Methods: Mice were immunized by intradermal injection of 1mg/ml peanut lectin. Fourteen days following the initiation of exposure, draining auricular lymph nodes were excised and a single cell suspension prepared. Draining lymph node cells from peanut primed or naive mice were labeled with carboxyfluoresein succinimidyl ester (CFSE) to identify proliferating cells, and restimulated in vitro with peanut lectin or with the T cell mitogen concanavalin A (con A) for various periods of time. Cells were stained with fluorescently-labeled anti-CD8 or -CD4 antibodies and following saponin permeabilization with fluorescently-labeled anti-cytokine antibodies.Results: In vitro stimulation of peanut-primed cells with peanut lectin or con A induced proliferation of CD4 and CD8 cells from 48-120 hrs. In contrast, cells from naRve mice responded only to con A. Moreover, allergen-specific CD4 cells expressed a Th2 profile with increased frequencies of IL-4 (6.3%) and IL-10 (5.9%) and relatively low levels of IFN-g (0.9%) positive cells compare with unstimulated controls or with cells cultured with con A, after 96 hrs in culture. CD8 cells displayed a Tc1 phenotype with high levels of IFN-g (6.5%) positive cells but few IL-4 (0.7%) and IL-10 (1.3%) positive cells regardless of whether restimulation was with con A or allergen.Conclusion: These data suggest that CD4, rather than CD8, T lymphocytes are skewed towards a selective type 2 cytokine phenotype in a mouse model of peanut allergy. TGFh is a highly conserved multifunctional cytokine that has diverse regulatory roles in the immune system. Regulatory T cells that secrete TGFh and variable amounts of IL-4 and IL-10, termed Th3, can be induced by oral administration of myelin proteins and mediate recovery from EAE. Little is known about the differentiation, phenotype and function of these cells. We created an inducible TGFh-1 transgenic (Tg) mouse model in which TGFh is linked to the IL-2 promoter and thus allows tissue specific expression of TGFh upon TCR stimulation. We found that antigen specific stimulation of naRve peripheral CD4+CD25-T cells from TGFh Tg mice induces Foxp3-expressing Th3 cells that are hyporesponsive and that have potent suppressive activities in vitro and in vivo. TGFh Tg cells do not secrete IL-2, IFN-g, IL-13 or IL-10. Early expression of TGFh, not IL-4 or IL-10, is critical for the differentiation of Th3 cells. In a MOG peptide TCR Tg adoptive transfer model, Th3 cells from TGFh Tg mice not only prevented the generation of pathogenic Th1 cells in wild type animals before the induction of EAE, but also greatly inhibited the effector functions if transferred at the time of disease onset. Using a two-step in vitro culture system, we found that antigen-presenting dendritic cells can act as dtemporal bridgesT to relay the inhibitory signal from Th3 cells to naive CD4 T cells. Furthermore, Th3 cells inhibit the T cell induced up-regulation of CD80/CD86 costimulatory signals during activation but not the maturation. Thus, the suppression by Th3 cells is mediated by mature dendritic cells with altered antigen-presenting function. Previously, we showed that treatment with the HMG-CoA reductase inhibitor Atorvastatin (AT) prevented the Th1 differentiation of myelin-reactive T cells and ameliorated clinical symptoms in mice with experimental autoimmune encephalomyelitis (EAE) (Youssef et al., Nature 420: 78, 2002) . HMG-CoA reductase is a critical enzyme in the mevalonate biosynthetic pathway that generates not only cholesterol, but also isoprenoid derivatives that function to attach certain signaling proteins and ubiquinone to cell membranes. The aim of the present study was to distinguish which of these pathway intermediates have a regulatory function in Th1 differentiation during the development of EAE. Here, we provide first-time evidence that oral administration of AT induces a Th2 bias CD4 + cells by compromising the production of the isoprenoid lipids farnesyl-pyrophosphate (-PP) and geranylgeranyl-PP. In vivo depletion of these metabolic precursors by AT caused a transient (i.e., 4-12 h/d) redistribution of farnesylated Ras and geranylgeranylated RhoA GTPases from the membrane to the cytosol of T cells. This was accompanied by a reduction in the phosphorylation of ERK and the DNA binding of c-fos in response to T cell receptor activation. We also show that selective inhibition of the ERK pathway with the MEK inhibitor PD98059 shifted the balance in T cell cytokine production towards Th2. Since ERK activation has been shown to be required for transactivation of IFN-g and for repression of the IL-4 promoter (Jorritsma et al., J. Immunol. 170: 2427 , 2003 , these results thus explain why CD4 + cells undergo Th2 differentiation when activated by antigen in the presence of AT. Chronic inflammation in rheumatoid arthritis (RA) is mediated by repeatedly activated pro-inflammatory Th1 cells. In contrast, Th2 cells that might down-modulate the chronic autoimmune response are rarely found in RA. It has been previously documented that RA T cells are severely impaired in their ability to differentiate into Th2 effectors while exerting enhanced Th1 differentiation. The mechanisms underlying this functional abnormality, however, have not been delineated. As interleukin-4 (IL-4) is a most critical determinant in regulating immune responses by promoting Th2 cell development and inhibiting Th1 cell differentiation, we analyzed the role of single nucleotide polymorphisms (SNP) in the IL-4 receptor a-chain, which is critical for binding of IL-4 and for IL-4 signal transduction, in the differentiation of human T cells. 361 healthy individuals were genotyped by allele specific PCR for the two IL-4R a-chain SNPs that are located in functionally important regions of the IL-4R a-chain-the I50V SNP50 and the Q551R SNP551 in the IL-4-binding and STAT6binding domains, respectively. Naive and memory CD4 positive T cells were isolated from the peripheral blood of individuals who were homozygous for either allele at SNP50 and SNP551, and primed for five days with mAbs to CD28 and/or CD3 in the presence or absence of exogenous IL-4. The phenotype of the resulting differentiated effector cells was then analyzed by flow cytometric analysis of cytoplasmic cytokines. The SNP551 alleles did not affect T cell differentiation. In contrast, the inhibitory effect of IL-4 on Th1 cell differentiation was significantly diminished in CD4 T cells that were homozygous for the mutant allele at SNP50 (50V) as compared to those with the wild type allele (I50). Likewise, the augmenting effect of IL-4 on Th2 cell differentiation was enhanced on T cells that were homozygous for the wild type allele as compared to T cells expressing the mutant allele. These data indicate that the mutant allele of the IL-4R achain at SNP50 is associated with a decreased T cell response to IL-4. To delineate a potential mechanism of different responses to IL-4 in the cells expressing different alleles of the IL-4R, T cells form individuals who were homozygous for either the wildtype or the mutant allele at SNP50 were primed with different concentrations of IL-4 and analyzed by flow cytometry for STAT6 and phosphorylated STAT6. Whereas STAT6 concentrations were not different between T cell expressing I50 or V50, STAT6 phosphorylation in response to IL-4 stimulation was significantly reduced in T cells expressing the V50 allele compared to T cells expressing I50. Thus, the V50 SNP50 allele of the IL-4R a-chain might regulate T cell differentiation by diminishing T cell responses to IL-4, resulting in reduced STAT-6 phosphorylation and subsequently in diminished Th2 cell differentiation. The V50 SNP50 allele might thereby contribute to the development of unbalanced Th subset activation, as characteristic for autoimmune diseases, such as RA. The IL-12 cytokine family (consisting of IL-12, IL-23, and IL-27) is proposed to mediate Th1 immune responses. Therefore, we utilized subunit-specific neutralizing monoclonal antibodies or genetic knockout mice to distinguish the individual contributions of IL-12, IL-23, and IL-27 in established murine models of Th1 autoimmunity and pathogen responses, namely experimental autoimmune encephalomyelitis (EAE) and Leishmania major infection. Specific neutralization of IL-12p35, or mice genetically deficient in IL-27-EBI3 demonstrated no protection from the incidence or severity of EAE. However, they each exhibited transient susceptibility to L. major infection as demonstrated by increased lesion size. In contrast, specific in vivo neutralization of IL-23 provided significant and long-lasting therapy of EAE when antibodies were administered prior to disease induction or onset, or during established EAE. Anti-IL-23 suppressed central nervous system inflammation and pathology even though antigen-specific proliferation and cytokine re-stimulation responses were not influenced by in vivo antibody treatment. FACS analysis demonstrated that in vivo IL-23 neutralization preserved a more naive (CD62L hi , CD45RB hi , CD69 lo ) CD4+ T cell phenotype. Thus, IL-23 appears to participate in the in vivo activation and/or trafficking of encephalitogenic T cells. Mice treated with IL-23 specific neutralizing antibodies maintained their protective T cell immunity to L. major infection. Overall, IL-12 and IL-27-EBI3 seem to be more closely related in function than IL-23. Further investigation will likely continue to delineate the roles of IL-12, IL-23, and IL-27 in autoimmune disease and pathogen immunity. Objective: Development of arthritis in the K/BxN mouse model is dependent on the induction of very high titers of antibodies (Abs) against the glycolytic enzyme glucose-6-phosphate isomerase, or GPI, promoted by CD4+ T cells expressing a transgeneencoded T cell receptor (TCR) specific for GPI. Our goal was to determine whether this unusually strong autoAb response, presumably reflecting unusually potent help, depends on T cell differentiation to the T helper (Th)1 or Th2 phenotype. The answer to this question might generate important insights into human arthritides, such as rheumatoid arthritis (RA), associated with the production of autoAbs. OR-116-Diverging Methods: The roles of cytokines known to control Th phenotype were investigated by introducing the interleukin (IL)-4 and IL-12p35 knockout mutations into the K/BxN model, and evaluating the impact of these deficiencies on clinical arthritis, autoAb production and T cell activation. The IL-4expressing cell-types in KBxN mice were revealed by crossing in a knock-in alteration resulting in green fluorescence protein (GFP) expression controlled by endogenous IL-4 gene regulatory elements. Transfer experiments permitted the identification of the IL-4-producing cell-type required for arthritis development. Finally, quantitative RT-PCR allowed determination of the cytokine profile of K/BxN T cells.Results: While IL-12p35 appeared dispensable for the development of arthritis, IL-4 was crucial for full disease development. IL-4-deficient K/BxN mice had greatly reduced titers of anti-GPI Ab. The GPI-reactive TCR of standard K/BxN mice induced the transcriptional activation of the IL-4 locus in CD4+ T cells and in CD11b+ eosinophils. Yet, K/BxN arthritis is not a pure Th2 disease, as both Th1-and Th2-type cytokines were upregulated in K/BxN T cells, and the expression pattern of several cytokines in K/BxN T cells did not match that of conventional Th2 cells.Conclusion: IL-4 is crucial for the development of anti-GPI-Abmediated arthritis in the K/BxN mouse model. However, the cytokine profile of initiating anti-GPI T cells does not fit that of a classical Th2 disease. The potential for IL-4 to promote the development of inflammatory arthritis should raise caution over proposed therapies for RA aimed at biasing T cells towards IL-4 production.OR-118. 1 1 Molecular Pharmacology and Physiology, Biogen Inc., Cambridge, MA, USA.Collagen-induced arthritis (CIA) is an inflammatory joint disease in rodents. Its etiology involves pathogenic autoimmune responses, which are provoked by immunization with collagen type II (CII) together with adjuvant. Mycobacteria, as part of complete FreundTs adjuvant (CFA), are potent inducers for IL-12 production by macrophages and DC. IL-12 is a key cytokine that instructs naive T cells, upon activation, to differentiate along the T helper type 1 (Th1)-pathway. CIA, like RA, claims to be regarded as a predominantly Th1-type autoimmune disease. However, as disease progresses, certain Th2-type features become detectable. It appears that CIA and perhaps RA are mixed type immune responses.A recently described heterodimeric cytokine, IL-23, shares the p40 subunit with IL-12. Both cytokines are implicated in either initiating or sustaining Th1-type responses. We have extended these studies and found that, provided that IL-12/23p40-deficient mice had been sensitized with pristane prior to priming with CII, severe CIA develops at 95% incidence.When pristane sensitization precedes CII/adjuvant immunization, mycobacteria become dispensable for CIA induction. CIA incidence and severity in wild-type mice immunized with CII is comparable to the disease observed in pristane-sensitized IL-12/ 23p40-K/O mice. Notebly, in wild-type mice, repetitive immunization with CFA can substitute for the requirement of pristanesensitization to maximize CIA severity. Taken together, the data suggest that pristane triggers an IL-12/23independent pathway capable of enhancing the auto-immunogenic stimulus set by CII. Finally, it appears that both, IL-12 and IL-23 are sufficient, but not necessary to trigger severe arthritis. Systemic Onset Juvenile Idiopathic Arthritis (SOJIA) remains an enigmatic pediatric rheumatic disease. Most patients require systemic corticosteroids for prolonged periods to control the systemic manifestations, and half the patients develop chronic arthritis that is difficult to control even with methotrexate and anti-TNF agents. The IL-1 antagonist Anakinra is partially effective in the treatment of inflammatory chronic arthritis, but it has not been evaluated in SOJIA patients with systemic symptoms. Immundiagnostic Disease Predictors We found that heat shock inhibits degranulation of BMMC without effects on leukotriene production. To further elucidate the mechanism of suppression of degranulation, we studied the effects of heat shock on calcium influx and tyrosine phosphorylation. We found that heat shock inhibits calcium influx and tyrosine phosphorylation of Syk and SHIP. Since degranulation of mast cells play a role in allergic and non-allergic reactions our finding may have a relevance with respect to protective effects of heat shock response. In addition to the conventional beffectorQ functions of eosinophils, evidence that eosinophils function as antigen-presenting cells (APCs) has been increasing. Amateur APCs stimulate only previously activated T cells and T cell hybridomas, whereas professional APCs are capable of initiating T cell responses. To investigate whether eosinophils are capable of initiating T cell responses in vivo, eosinophils were isolated from the spleens of IL-5 transgenic BALB/c mice by Percoll following MACS, and contamination with other APCs including dendritic cells was excluded. Co-culture of eosinophils with GM-CSF increased their expression of costimulatory molecules including MHC-II. The GM-CSF stimulated eosinophils were allowed to take up OVA in vitro and then intratracheally injected into wild-type BALB/c mice that received intravenous injection of Ag-specific CD4+ T cells from DO11.10 OVA TCR transgenic BALB/c mice 24 h earlier. By alternatively using GFP-labeled eosinophils from IL-5 & GFP double transgenic mice and fluorescently conjugated OVAbeads, we demonstrated by fluorescence microscopy that the Agloaded eosinophil APCs were physically interacting with naive OVA TCR CD4+ T cells in the draining paratracheal lymph nodes (PLNs) 24 h after eosinophil transfer, while Ag-free eosinophils were randomly distributed across the PLNs with the donor CD4+ T cells. The physical interaction between Ag-loaded eosinophils and Ag-specific CD4+ T cells resulted in the activation of the naive CD4+ T cells, as measured by an early T cell activation marker CD69 by flow cytometry. However, this eosinophil APC function was completely impaired if eosinophils were pre-treated with RBC lysis buffer containing ammonium chloride, which inhibits antigen processing by eosinophils. Our data suggest that eosinophils may function as professional APCs to initiate T cell responses to a given antigen. Background: TGFh-1, a multifunctional cytokine, has been shown to suppress immunoglobulin production in animal experiments. Plasma TGFh 1 has been observed to be higher in some asthmatics compared to normal controls (Joseph et al, Ann of asthma Allergy). The effect of TGF h-1 on cytokine production by human peripheral blood mononuclear cells (PBMC) has not been investigated. F1 Methods: PBMC from asthmatics and normal controls were isolated from heparinized blood by density-gradient centrifugation on Ficoll-Paque, washed three times in phosphate buffered saline and resuspended at 1 Â 106 cells/ml in serum free medium (AIM-V). For cytokine measurements, 1Â 106 PBMC/ml culture medium were incubated with phorbol-12-myristate 13-acetate (5ng/ml) and Ca2+ ionophore (0.4mg/ml) in the presence of TGF h-1 (100pg/ml) alone and with antibody to TGF h-1. Following 72 hours of cell culture, supernatants were collected and stored at -800C until assayed for cytokines. ELISA kits from Pharmingen, USA were used to determine IFN-g in the supernatants from PBMC cultures.Results: So far results from 4 controls and 6 patients have been analyzed. The median IFN-g production in unstimulated cells (5.5 pg/ml) increased significantly following exposure to TGF h-1 (52 pg/ml) (p b 0.01). In the resting state, there was trend for PBMC from asthmatics to produce higher amount of IFN-g compared to PBMC from controls.Conclusion: TGF h-1 may up regulate the production of IFN-g by resting and stimulated PBMC in normal controls and asthmatics and this response was abrogated by specific antibody to TGF h-1.The aim of the present study was to compare the clinical efficacy of rhinophototherapy with fexofenadine hydrocloride. We performed an open study on 18 ragweed-allergic patients, during the ragweed season in Szeged. 11 patients received intranasal irradiation with increaseing doses of mUV/VIS light for 2 weeks and 7 patients received 120 mg fexofenadine HCl once daily for the same period of time. Individual symptom scores and total nasal score (TNS) were recorded.Rhinophototherapy resulted in a significantly better reduction of individual symptom scores for rhinorrea (P = 0.0007) and nasal obstruction (P = 0.014) and of TNS (P = 0.004) compared with fexofenadine HCl. No significant differencies between the two treatments were observed in reducing symptom scores for sneezing, nasal itching, palate itching and eye symptoms. In addition, we have measured the wheal formation in skin prick test (SPT) by digital planimetry before starting the study and 10 days after ending the therapy. Interestingly, ten days after the end of the treatment, in the rhinophototherapy group the allergen-induced wheal formation was significantly reduced compared to baseline (P = 0.03), in contrast in the fexofenadine treated group no differences were observed. No changes in histamine-induced wheal formation were observed. In our study, rhinophototherapy was significantly more effective than fexofenadine in treating allergic rhinitis. The prolonged inhibitory effect of rhinophototherapy on SPT suggests a long lasting effect that was not seen after fexofenadine treatment. Eosinophils (Eos) are prominent cells in asthmatic inflammation. Once in the lung or airways, Eos show significantly prolonged survival. Anti-apoptotic activity is mediated by cytokines such as GM-CSF and IL-5, which are markedly increased in the asthmatic lung. Selective induction of Eos apoptosis has been proposed as a therapeutic approach for asthma. Previous studies have shown PPIase (cyclophilin A and FKBP) inhibitors suppress GM-CSF, IL-3 and TNF-a expression and function. These data implicate PPIase mediated cis/ trans isomerization of pSer/pThr-Pro bonds in target proteins as a potential key regulator of cytokine expression. Recently, we have shown that inhibition of Pin-1, another PPIase, blocked the pro-survival effect of either GM-CSF or hyaluronic acid (HA). To identify the mechanisms underlying Eos apoptosis induced by Pin1 inhibition, we examined caspase-3 (Casp-3) activation. Eos were treated with the Pin-1 inhibitor juglone at 1.0 AM and cell lysates examined for full-length Casp-3 proenzyme (p32) and active Casp-3 (p17) subunits by western blot analysis. As shown in previously published data, resting Eos underwent spontaneous (baseline) Casp-3 activation after 24 h in culture that was completely blocked by rhGM-CSF (100 pg/ml). Treatment of Eos with HA (100 Ag/ml), which is markedly increased in the airways of asthmatic lung, prevented spontaneous Casp-3 activation as well. However, treatment of Eos with juglone (1.0 AM) induced Casp-3 activation, even in the presence of rhGM-CSF or HA. Furthermore, apoptotic initiation by Pin1 inhibition was more apparent on Eos pre-activated with rhGM-CSF. Pre-incubation with high concentrations of rhGM-CSF or HA also failed to block juglone induced Casp-3 activation and cell death. Kinetic analysis showed that within 10 minutes juglone triggered extremely intense Casp-3 activation. Casp-3 activation was a very sensitive and early marker for the ultimate apoptosis of Eos. Trypan blue exclusion indicated that Eos viability remained high (between 86-97% at 4, 10 and 24 h) despite juglone treatment. These data indicate that Pin1 enzymatic activity is required for preventing Casp-3 activation and the initiation of apoptosis and does so downstream of the GM-CSF receptor. Objectives: Define in a BALB/c mouse in vitro model the dominant T cell epitopes of the major cat allergen Fel d 1. Test the effect of natural CD25 + CD4 + regulatory cells (T regs) on the response of CD25-CD4 + cells from naive mice and mice immunized with Fel d 1. Materials and Methods: For the analysis of the proliferative immune response of naive mice and immunized mice, we used an in vitro system where myeloidderived antigen-pulsed dendritic cells (DC) induced T cell proliferation. Immature DCs were harvested from mouse bonemarrow and matured in vitro for 7 days before being pulsed with antigen for 2 days. Lymphocytes were obtained from mouse spleen cells and added to the DC cells for 4 days, before 3 H thymidine addition and harvesting. Cell proliferation was measured for un-separated spleen lymphocytes and separated T cell subpopulations. CD25 + CD4 + and CD25-CD4 + T cells were separated by magnetic beads and checked for purity by FACS. T cell stimulation was measured with whole Fel d 1 and 17 overlapping peptides. Immune BALB/c mice had been injected 3 times with Fel d 1 in Al(OH) 3 . Results: Un-separated splenic lymphocytes from naive mice did not give a significant proliferation when stimulated by Fel d 1 allergen or the 17 synthetic peptides. Un-separated lymphocytes from immunized mice gave significant stimulation indexes with Fel d 1 and peptide F 1.4 (aa 20-40 on chain 1). Purified CD25-CD4 + lymphocytes from Fel d 1-immunized mice gave a significant stimulation with Fel d 1 and peptide F 1.4. When CD25 + CD4 + T regs were added to the CD25-CD4 + cells, proliferation was inhibited. Purified CD25-CD4 + cells from naive mice gave also a positive stimulation index when exposed to Fel d 1 or F 1.4 peptide. This proliferation was abolished by the addition of T regs from naive mice at a ratio of 1 T reg cell to 2 CD25-CD4 + cells. Increased Experession in CD30+ and CD57+ Molecules on CD4+ T Cells in Atopic Asthmatic Children: A Preliminary Report.N. 2 1 Clinical Immunology and Allergy, ISSSTE, Mexico, Mexico City, Mexico; 2 Biochemistry, INER, SSA, Mexico, Mexico City, Mexico.Background: The phenotype of CD4+ T cells accumulated in chronically inflamed tissue, in allergic process, have been found to be mainly CD4+ memory T cells that express surface markers associated with IL-4 production. However the process of these cells and surface markers in peripheral blood have been not clearly determined. Objective: In this study we performed the frecuency of surface markers on CD4+ T cells with IL-4 production in peripheral blood of atopic asthmatic children. The proportion of the peripheral mononuclear cells and surface molecules was studied by flow cytometry to identify surface molecules in CD4+ T cells (CD30, CD57, CD154, CD62L, and CD28), and IL-4. The analysis was performed on PBMC after PMA-Ionomycin stimulation, to examine IL-4 and INF-g production. Results: CD4+ CD30+ (median; 1.7, percentiles 25-75; 1.3-2.2) , and CD4+ CD57+ (median; 3.3, percentiles 25-75; 2.2-4.4) T cells showed an increased production and correlationship with IL-4 production in atopic asthmatic children. Conclusion: Although CD4+CD30+ T cells in peripheral blood have been observed in atopic dermatitis patients, in this work we identified similar cellular population in respiratory atopic diseases, and also CD57+ T cells, these cells seems to corresponds of CD4 T cells which expressing IL-4 under stimuli. That expressing markers could correspond early activation in atopic asthma. In allergic process a number of studies have analysed the phenotype of CD4+ T cells that express surface markers preferentially associated with IL-4 production. However the repercussions of nasal challenge over these cells in peripheral blood have been not clearly determinated. We found that CCR3 on CD4+ T cells correlated positively with IL-4 production. In conclusion the CD4 T house dust mite primed cells in allergic rhinitis patients expressing CCR3 that correlates with IL-4 production, these local challenge repercussion in peripheral CD4+ T cells could be observed only when those cells are stimulated with PMA-I. Mugwort pollen allergens represent the main cause of pollinosis in late summer in Europe. Ninety-five percent of mugwort-allergic patients are sensitized to the major allergen Art v 1. In contrast to other common pollen allergens which contain multiple T cell epitopes, Art v 1 contains only one single immunodominant T cell epitope (Art v 1 25-36 ). We characterized the minimal epitope of Art v 1 25-36 and investigated a possible association of Art v 1-reactivity with HLA class II-phenotypes.Art v 1-specific T cell lines and clones were established from 51 patients with clinically defined mugwort pollen allergy and IgE specific for Art v 1. In 96% of the patients a cellular response to Art v 1 25-36 was obtained and a core region of 5-10 amino acids containing 3-5 amino acids essential for T cell reactivity was defined by using truncated and single-substitution analog peptides for T cell stimulation. HLA-DRB1*01 was identified as the main restriction element for the presentation of the immunodominant epitope using monoclonal anti-HLA antibodies and APC with defined HLA-DRB and DQB1-alleles.In conclusion, allergy to Art v 1 is characterized by a uniform T cell reponse and the disease is associated with the HLA-DRB1*01phenotype. Therefore, mugwort pollinosis represents an ideal candidate for a peptide-based immunotherapy including the possibility of monitoring antigen-specific T cell responses during therapy by using HLA-DR-tetramers. Characterization of Human Cord Blood-Derived In Vitro Generated Mast Cells: Hemopoietic Antigens, Chemokine Receptors, Activation Markers, Tetraspanins.I. Mirkina, T. Schweighoffer. 1 RD-ADV, Novartis Institutes for Biomedical Research, Vienna, Austria.Mast cells (MC) play pathogenic role in allergic inflammation via releasing a broad spectrum of inflammatory mediators. We generated MC from human cord blood CD133+ hemopoietic precursors by culturing with rhSCF, rhIL-6 and rhIL-3 in Stem Span medium. To better characterize differentiation process, we mapped the expression of hemopoietic markers and chemokine receptors. Adhesion molecule ICAM-1 (CD54), IL-3 receptor (CD123), aminopeptidase N (CD13) and CD38 were present on MC and their precursors. Early hemopoietic markers CD133 and CD34, bright on freshly isolated precursors, disappeared within 2 weeks of differentiation. Development into mature MC was enhanced when cells underwent freezing/thawing cycle followed by culturing in the presence of 5% human serum. After 5 to 7 weeks they displayed typical features of mature MC: methachromatic staining with Gimsa-May Gruenwald, abundant expression of granular mast cell tryptase; surface expression of MC antigens c-kit (CD117) and FceRIa; degranulation after cross-linking FceRIa by IgE (+Ag). Both MC and precursors markedly expressed surface CXCR2 and CXCR4 and were negative for CCR3. Interestingly, we detected chemoattractant receptor homologous molecule expressed on Th2 cells, CRTH2, on the surface of MC and their precursors. As CRTH2 is a second receptor for prostaglandin D2 (PGD2), and PGD2 is a major prostanoid released from Agactivated MC, our data suggest possible autocrine function of PGD2 for MC.To find sensitive marker(s) of MC reactivity to Ag, we explored the correlation between MC degranulation and expression of activation markers CD63 (tetraspanin) and CD203c, both used for testing reactivity of basophils to allergens. We found both markers to be hardly detectable on the surface of MC precursors but high on mature MC both at the surface and intracellularly. This is the first evidence of CD203c presence on cord blood-derived MC. Expression of both CD63 and CD203c was further increased after IgEdependent and independent stimulation, and this increase mirrored degranulation process.We determined other members of tetraspanin family, CD9 and CD81, to be high on the surface of MC precursors. Expression of CD9 and CD81 was further augmented up to 10 fold with differentiation to mature MC. In contrast to CD63, surface expression of CD9 and CD81 diminished after stimulation with PMA/ionomycin but not after triggering with IgE (+Ag). Therefore, members of btetraspanin webQ could be differentially involved in MC activation.These studies define potential targets for anti-allergic intervention and sensitive tools to monitor MC activation. Rationale: ABPA is a Th2 hypersensitivity lung disease resulting from bronchial colonization by Aspergillus fumigatus in asthmatic and cystic fibrosis patients. We propose that single nucleotide polymorphisms (SNPs) of IL-4Ra also play a role in the development of ABPA in asthmatic and CF patients.Methods: DNA was extracted from cultures of B-cell lines of 26 ABPA and 29 non-ABPA patients and sequenced for IL-4Ra polymorphisms, including 1 extracellular (ile75val) and 4 cytoplasmic (glu400ala, cys431arg, ser503pro, and gln576arg) SNPs. IL-4 stimulated PBL from ABPA and control subjects were examined for the expression of CD23 on B cells by flow cytometry. HLA-DR genotyping was performed using standards techniques.Results: The frequency of IL-4Ra SNPs was significantly increased in ABPA patients compared to non-ABPA subjects, 92% vs 55%. Cytoplasmic SNPs were identified in 39% of the ABPA patients, and co-existence of extracellular plus cytoplasmic SNPs were observed in 27% of ABPA patients. ABPA subjects also had significantly increased expression of CD23 molecules per B cell of IL-4 stimulated PBL cultures compared to controls. In one ABPA patient, the asn98thr SNP was associated with ile75val and ser503pro SNPs, and in the other patient the asn98thr SNP was isolated. This was also associated with up-regulation of CD23 expression on B cells by IL-4 stimulation.Conclusions: The presence of IL-4Ra SNPs, particularly ile75val allele located within the IL-4 binding region may confer susceptibility to developing ABPA. Background: House dust mite(HDM) allergen are involved in sensitization and development of allergic airway disease, particularly bronchial asthma and allergic rhinitis. Dermatophagoides pteronyssinus(Dp) and Blomia tropicalis(Bt) are the predominant inhalant allergens in most parts of the world. Aim: to measure Derp1 and Blot5 allergen levels in asthmaticsT homes in HongKong. Methods: Seventy houses were enrolled for a mite indoor environment study. Dust samples were obtained from two sites of each subjectsT house: bed and floor. Derp1 and Blot5 levels were quantified by a two-site monoclonal antibody-based ELISA techniques. Results: The levels of Derp1 allergens were found in bed (GM: 3.43 ug/g of dust; 95%CI: 1.89-4.96 ug/g) and on floor (GM: 1.12 ug/g of dust; 95%CI: 0.71-1.53 ug/g) with significant differences(P = 0.005). However, the levels of Blot5 allergens were also found in bed (GM: 19 ug/g of dust; 95%CI: 0.89-38.9 ug/g) and on floor (GM: 6.14 ug/g of dust; 95%CI: 0.4-11.9 ug/g), with no statistically significant difference Blot5 allergens found in the different sites. In addition, Concerning the exposure index for Derp1 and Blot5 allergens found in bed and on floor, 17.6% in bed and 8.6% on floor had levels of Blot5N=10 ug/g of dust, higher than those obtained for Derp1 (7.2% and 0% in bed and on floor respectively, p b 0.05); On the other hand, higher percentages in bed and on floor (25% and 35.7%)were observed for the levels of Blot5=0ug/g of dust as compared with Derp1 in bed and on floor (4.3% and 14.5% respectively, p b 0.05). Conclusions: Der p1 and Blot 5 are the major sensitizing allergens in this region, Blot 5 is a more potent one in HongKong, probably reflecting the high level of exposure to Bt. The unique major Bt and Dp allergens should be included for precise diagnosis and effective immuno-therapeutic treatment of mite allergy in HongKong. cytokines in bronchial epithelial cells. Objectives: we have investigated whether Der f allergen proteases induced cytokine production from the epithelial cell line BEAS-2B. Methods: Cells were exposed to four different concentrations with serial additions of Der f (0.02, 0.2, 2, 20 ug/ml) were incubated for 24 h to 96 h. and compare with those without incubation of allergen. Cytokine in the supernatants were assayed by ELISA, Reverse transcription-PCR was also performed. Results: Cells treated with Der f allergen showed serial changes in the cohesiveness of the monolayer. There was a significant increase in the level of cytokine production compared with the untreaed sample. Statistically Significantly increased with addition of Der f caused the release of IL-6 and IL-8 in time and concentration-dependent manner (p b 0.05,respectively). Levels of IL-6 and IL-8 were elevated 24 h and 48 h after allergen exposure, increasing with time, continued increased levels to be present of IL-6 and IL-8 in the supernatants at 72 h and 96 h. At the same time show the concentration dependence of induction of IL-6 and IL-8 expression as well as an increase in the expression of IL-6 and IL-8mRNA. Conclusion: HDM-induced airway inflammation may include Der f-mediated release of inflammatory mediators, and the proteolytic activity of an allergen may stimulate the release of proinflammatory cytokines from human bronchial epithelium. Suggesting that IL-6 and IL-8 production by bronchial epithelial cells may play a role in the pathogenesis of allergic asthma. Introduction: The pathogenesis of airway remodeling involves altered interactions between epithelial and mesenchymal cells that lead to air wall thickening and edema (Davies D. et al., J. Allergy Clin. Immunol., 2002; Frieri M. Allergy Asthma Proc. Airway remodeling is associated with increased VEGF and increased vascular permeability in the pulmonary submucosa (Lee KS et al., J. Allergy Clin. 300, 600 and 1000 AU/ml dialyzed D. pteronyssinus extract (DpE) stimulated cA549 to secrete VEGF into serum-free conditioned media (CM) relative to DpE without cA549 (control media; CTLM) in 24 hours (Capetandes A. et al., Am. Rationale: Determine if mediators secreted into the CM by DpE-treated cA549 can stimulate NHLF to secrete VEGF relative to CTLM. Methods: Subconfluent NHLF were cultured for 24 hours with CM and CTLM generated with and without cA549, respectively, plus 0, 300, 600 and 1000 AU/ml DpE. The CM and CTLM were assayed for VEGF by ELISA. Cell number was measured by MTT incorporation at A550. Results: NHLF in serum-free media secreted VEGF relative to control (181 F 23 pg/ml (n = 6) versus 3.3 F 0.5 pg/ml (n = 4); P b 0.0001, t test); CTLM did not stimulate NHLF secretion of VEGF over control (n = 6). Absolute VEGF levels were increased by the following conditions: CM + NHLF (1017 F 97 pg/ml (n = 12)) N CM w/o NHLF (611 F 44 pg/ml (n = 4)) N CTLM +NHLF (198 F 38 (n = 12) ; P b 0.0001 ANOVA). 1000 CTLM caused a decrease in NHLF cell number relative to CM 1000 ( This study was performed to evaluate the probable effect of allergy in children with and without otitis media with effusion (OME). Is allergy more common in OME patients? Allergic patients might be helped with allergy treatment. Otitis media with effusion, a common childhood ear disease, has various predisposing factors that one of them may be allergy. Skin prick test (SPT) is able to determine allergic patients to common aeroallergens. The study was performed on 30 children with OME in khalili Hospital, a teaching hospital affiliated to Shiraz University of Medical Sciences in Iran. Myringotomy with or without ventilation tube insertion plus adenoidectomy were done for them. A group of 30 pateints in the same age range (2-9 years) with normal middle ear whom underwent adenoidectomy was selected as control. Skin prick test for common aeroallergen (molds, grasses, weeds, trees and mites) was done. The presence of peripheral eosinophilia was also investigated in both groups. Peripheral eosinophil counts were significantly higher in the case group (p b 0.01). SPT was negative in all children in the control group, however skin reactivity between two groups was not significantly different. We were not able to demonstrate a strong correlation between OME and positive skin test to common aeroallergens. D. Micheloud, 1 J. Jensen, 1 E. Fernandez-Cruz, 1 J. Carbone. 1 1 Clinical Immunology Unit, University Hospital Gregorio Maranon, Madrid, Spain. There have been no reported cases of angioedema by Blastocystis hominis. Materials: Clinical and immunological data of a patient with such association. Case report: The subject was a 21-year-old female with a 5-years history of episodic attacks of swelling of mouth, face and upper extremities accompanied by recurrent urticaria. She had been treated with different antihistamines and oral corticosteroids with only a partial response. Inmunological tests performed in peripheral blood disclosed normal immunoglobulin levels (IgG 1160 mg/dl, IgA 182 mg/dl, IgM 120 mg/dl), normal percentages of lymphocyte subsets (CD3 77%, CD4 55%, CD8 18%, CD19 12%, CD56 7%); normal level of the complement factors C4 (19 mg/dl), C3 (100 mg/dl), FB (28 mg/dl) and of C1-inhibitor (15 mg/dl) as well as negative circulating immune complexes. IgE specific to ascaris, echinococcus and anisakis were negative. Serologies for hepatitis virus B, hepatitis virus C, and HIV were negative. Complete blood and differential analysis as biochemical serum parameters were around normal ranges. Stool examination revealed Blastocystis hominis at 3 consecutive determinations. Remission of urticaria-angioedema have been maintained after a 24-month of clinical follow-up. Conclusion: Diagnosis of Blastocystis hominis infection must be suspected in patients with otherwise frustrating chronic allergic skin disorder. Paromomycin might be of benefit in chronic persistent urticaria-angioedema associated with this parasitic infection. Two groups recently have reported that various mouse monoclonal IgE antibodies can induce mouse bone marrow derived mast cells to secrete mediators in the absence of known specific antigen. In this study, we investigated whether exposure to purified human myeloma IgE (catalog number A12162H, Biodesign International, Kennebunk, ME) in the absence of known specific antigen had detectable effects on the mediator secretion of human mast cells that were generated in vitro from umbilical cord blood cells. Exposure to IgE at 2.5 micrograms/ml, but not IgG, significantly enhanced the release of chemokines, but not histamine or cysteinyl leukotrienes, from human mast cells. These results were obtained both with microcentrifuged preparations of IgE (which lacked large aggregates of IgE according to HPLC and mass spectrometry) and with HPLC-purified preparations of IgE monomers that were devoid of IgE dimers according to mass spectrometry. However, under all conditions of challenge tested, chemokine production in response to IgE alone was significantly less than that induced when aliquots of the same IgEsensitized populations of human mast cells were stimulated by anti-IgE. The production of chemokines in response to exposure to IgE in the presence or absence of anti-IgE was inhibited by preincubation of the cells with dexamethasone. Overall, these results indicate that exposure to human myeloma IgE in vitro in the absence of known specific antigen can induce chemokine production by human mast cells at the concentrations tested. While the clinical relevance of these findings remain to be determined, one might speculate that effects of IgE on mast cells that are independent of known specific antigen can contribute to the pathogenesis of mast cell-associated disorders, particularly in subjects with high levels of IgE. We have recently showed that intranasal phototherapy using mixed low dose UVB, UVA and visible light (mUV/VIS) is effective in treating seasonal allergic rhinitis. Conclusions: The major cat allergen Fel d 1 seems to harbour one major T cell epitope containing region when tested in immunized BALB/c mice. Natural T regs from immunized mice inhibit CD25-CD4 + lymphocyte proliferation, when stimulated by Fel d 1-allergen or F 1.4-pulsed DC. Natural T regs from naive mice inhibit CD25-CD4 + proliferation from naive and immunized BALB/c mice when tested with Fel d 1-and F 1.4 peptidepulsed dendritic cells. Background: Omalizumab (OMA) is a novel humanized monoclonal anti-IgE antibody for allergic asthma. OMA binds circulating IgE, leading to a reduction in high affinity IgE receptors on mast cells (MC), thereby reducing MC degranulation upon specific allergen exposure. This results in a decrease in MC release of allergic mediators such as histamine and leukotrienes (LTs). LTRAs block the effects of cysteinyl LTs responsible for some features of allergic asthma, however, they do not block other mediators or categories of LTs released by MC. To examine the effect of OMA in moderate-severe allergic asthma in patients using LTRAs, we evaluated asthma exacerbations and need for bursts of systemic steroids in a pooled analysis of two recently completed clinical trials.Methods: INNOVATE (a 28 week randomized double-blind placebo-controlled study) and ETOPA (a 52 week open label trial) allowed concurrent LTRA use and were used for this analysis. Entry criteria and clinical outcomes were similar allowing for a pooled analysis. A total of 731 patients were in the intent-to-treat (ITT) population in the two studies. All patients received inhaled steroids (mediandose2000AgBDPequivalent).Longactingbetaagonistswere used by all patients in INNOVATE and 87% of patients in ETOPA receiving concurrent LTRAs. Overall, LTRAs were used at baseline in 32.3% of patients (INNOVATE 34.8%, ETOPA 28.9%). Groups were compared using Poisson regression based on the ITT population, adjusting for baseline sex, age, use of oral steroids, FEV1 (N=80%, 60-b80%, b60%), study treatment and treatment-by-LTRA interaction.Results: Patients on LTRAs tended to exhibit a greater level of asthma severity as evidenced by baseline history and trial incidence of clinical exacerbations, irrespective of treatment. The relative risk (RR) of asthma exacerbations (primary outcome; OMA vs. control) of the LTRA subgroup was 0.62 (95% CI: 0.42-0.91), which was somewhat lower than that observed for the overall study population. Similarly, the RR for use of systemic steroids (secondary outcome; OMA vs. control) for the LTRA subgroup was 0.5 (95% CI: 0.35, 0.72), similar to the effect size observed for the overall population.Conclusions: In patients with moderate to severe asthma, OMA demonstrated efficacy in the LTRA subgroup that was similar to improvements shown in the overall population. Asthma morbidity, as assessed by clinical exacerbations, was improved in conjunction with significant reductions of systemic steroid bursts. Inflammation in allergic asthma is generated and activated by endogenous proinflammatory cytokines including IL-4 and IL-5 produced by Th2-type lymphocytes. These allergen-induced Th2 responses enhance airway hyperreactivity in mouse models. In this study, we have shown that development of Th1/cell-mediated immune response significantly down-regulated Th2 responses by eliciting IFN-g production in experimental induced ova albumin (OVA) allergic BALB/c mice. Inhalation of chitin was made or mice were given chitin intravenously during the OVA-sensitization. Allergen-induced immunopathological responses, such as BALF cytology, anti-OVA humoral responses, and OVA-driven cytokines production were assessed. To dissect the inhibitory mechanisms of Th2 responses, spleen cells isolated from the chitin-treated or nontreated OVA-sensitized mice were cultured in the presence of OVA and/or chitin for 5 days. OVA alone stimulated the production of Th2 associated cytokines in both groups; in contrast, OVA/chitin stimulation resulted in the significantly increased production of IFN-g. Moreover, spleen cells isolated from the chitin-treated mice showed abundant amounts of IFN-g production with the stimulation of chitin, and less amounts of Th2 cytokine with or without OVA-stimulation, suggesting that the inhibited Th2 responses might explain the potential mechanisms, due to the changes in antibody isotypes and cytokines produced from splenocytes of mice receiving chitin. In summary, these results indicate that chitin-induced IFN-g responses successfully down-regulate Th2facilitated IgE production and lung eosinophilia in the OVA allergic animals. Purpose: The idea is to enlighten the series of allergic/ toxic manifestation with an un-expected onset on ingestion of cooked mushroom (Cortinarius orellanus), Gyromitra. Gyromitra, poisonings have also occurred after ingestion of commercially available morels contaminated with G. esculenta.Methods: 10 cases (ages 16-50-years both sex) had been notified for treatment (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) to the emergency department of Al-Junaid hospital, with ingestion of cooked mushroom as vegetable (toxic species are confused with edible species), followed by adverse reactions (allergic/toxic) with a variable severity.Allergic manifestations i.e., Itching burning flushing, tingling sensations, all over the body, Urticaria with variable severity.Toxic Manifestations: Were Acute in onset(resulting from neurotoxins release) i.e., Nausea (15-30 min), vomiting (20-60 min) abdominal cramping, bloated feeling. watery diarrhea (20 min-13 h), prosteration,dehydration, profuse sweating, coma convulsions hallucinations, excitement, depression, spastic colon, seizures (20 min-13 h), extreme thirst, and lack of urine production, Other symptoms included feeling of warmth, clamminess, numbness of the tongue and extreme thirst.One case in series had concomitant intake of alcoholic beverage with toxic syndrome. clinical testing procedure i.e, using a 3Hradioimmunoassay (RIA) test kit had evidenced sub-nanogram levels of toxin in urine and plasma. 4/10 Patients survived this early phase& recovered without any complications with meticulous follow up, 6/10 with much severe manifestations/delayed in notification for treatment appeared to have recovered for a short time, but this period was generally followed by a rapid and severe loss of strength, prostration, and pain-causing restlessness, sudden onset of abdominal discomfort (a feeling of fullness). Aggressive therapy resulted in survival 4/10 (out of 6/10) with variable degree of liver enlargement. The rest of 2/10 of (6/10) succumbed to death from irreversible damage to vital organs(hepato-renal insufficiency, cardiac, and skeletal muscle). The toxin affected primarily the liver, but there are additional disturbances to blood cells and the central nervous system.Results: The degree of reversal of adverse effects depends upon the urgency of therapeutic notification. In the absence of dietary history, Allergic/toxic manifestations could be mistaken for symptoms of hepatic renal impairment as a consequence of other causes (e.g., viral hepatitis), therefore an urgent distinction be made, as the delayed onset of symptoms will be mistaken behind the idea that the organs have previously been damaged. The importance of rapid diagnosis is evident, victims who are hospitalized and given aggressive supportive therapy immediately after ingestion have a mortality rate of only 5-10%, whereas those admitted 50hours or beyond after ingestion have a 55-85% mortality rate.Conclusions: Mushrooms as per its names are alike,but dissimilar in their nutritional & toxicity nature, its haphazard selection/consumption could cost life of the consumer. Tacrolimus is a macrolide immunosuppressant used to prevent graft rejection in transplant patients and in the treatment of atopic dermatitis. The drug inhibits T-lymphocyte activation by preventing the transcription of IL-2. We wish to investigate whether tacrolimus also indirectly suppresses natural killer (NK) cell and eosinophil activation by inhibiting T-cells. Lymphocyte Assay: Heparinized blood was collected from healthy adult donors and was layered over a ficol-hypaque density gradient to isolate lymphocytes. Cells were cultured in-vitro at a concentration of 1.0 Â 10 6 cells/ml in IL-2 and increasing amounts of tacrolimus. The drug and cytokines were added bi-weekly to maintain the appropriate cell concentration. Weekly flow cytometric analyses were performed to detect tacrolimusT effects on T-helper cell (CD4+), T-cytotoxic cell (CD8+), and NK cell (CD16+ and CD 56+) populations. CD 69+ was also measured to assess lymphocyte activation by double staining for CD 4+/69+ and CD 8+/16+. Weekly assessments of 51 Cr discharges from K562 cells were performed to detect NK cell activity. Eosinophil Assay: Tacrolimus effect on eosinophil viability was investigated by isolating white blood cells from eosinophilia patients. Whole blood collected from patients was layered over a 75% percoll gradient. The isolated white blood cells were kept at a concentration of 1.0 Â 10 6 cells/ml and were treated with IL-5. Varying amounts of drug concentration were added to specific cultures in order to study its differing effects on cell activation. Cytokine and drug were added biweekly and flow cytometry was performed at 4, 7, and 14 day increments to monitor eosinophil activation through CD69+ expression and fluorescence. Lymphocyte Activation: Inhibition was observed in T-lymphocyte and eosinophil populations as well as NK cell activity. Flow cytometry analysis staining for CD4+/69+ expressions indicate that with increasing time and concentration of tacrolimus, T-lymphocyte activation decreases. At week 2, there was an 8.3% and 14.2 % decrease in CD4+/69+ activation levels with tacrolimus 50 ng/ml and 500ng/ml respectively. CD8+ overall activation level was unaffected with increasing treatment of drug and time, while 51 Cr assay suggested an overall decrease in NK cell activity. Eosinophil Activation: Compared to control, flow cytometry results staining for CD 18+/ 69+ indicate a 9% and 6% decrease in eosinophil activation at 4 days of incubation. At 14 days, there was a 19% and 23% decrease in cell activation with tacrolimus 50 ng/ml and 500 ng/ml. With increasing time and concentration, mean fluorescence for cells expressing the CD 69+ activation marker decreased. Conclusion: Although the primary effect of tacrolimus is on T-cells, it also may affect NK cell and eosinophil activation. The effect on eosinophils may explain the drugTs beneficial effect in atopic dermatitis patients. Hemopoiesis is an important factor in the pathogenesis of allergic asthma. Several studies suggest that extramedullary hemopoietic cells present inside the asthmatic lungs contribute to chronic airway inflammation. To define the factors responsible for the emergence of these cells, and their relationship to allergic inflammation, we isolated intrapulmonary hemopoietic cells from the lungs of allergic BALB/c mice, and showed that: a) their presence is strictly dependent on airway challenge of ovalbumin-sensitized mice (Chest, 2003, 123, 345S) ; b) they differ from hemopoietic cells in bone-marrow in their growth properties and sensitivity to steroids (Intl. Immunopharmacol., 2005, in press ). Here we evaluated the possible contribution to intrapulmonary hemopoietic cell accumulation made by systemically active signals originating in challenged lungs, and by the local allergic reaction. To define: a) whether allergen-challenged lungs release factors responsible for intrapulmonary accumulation of hemopoietic cells; b) whether the production and activity of these factors can be dissociated from allergen-induced lung injury. Methods.We developed a transplantation model in which fragments of allergen-challenged, sensitized lung donors were ectopically implanted in syngeneic recipients, and hemopoietic cells inside the recipientsT lungs were quantitated without allergen exposure of the recipients. In BALB/c mice, accumulation of hemopoietic cells occurred only when: a) donors were sensitized and challenged in the airways; b) recipients were sensitized through 2 sc allergen injections, but not airway-challenged.Media conditioned by lung fragments from the appropriate donors contained biologically active IL-5, as well as immunoreactive IL-5 and eotaxin, and induced intrapulmonary accumulation of hemopoietic cells in sensitized recipients. Unlike BALB/c, lungs from IL-5 transgenic CBA/Ca mice contained a large number of hemopoietic cells, independently of sensitization and challenge. Lung fragments from naive, IL-5 transgenic donors (or their conditioned media), induced intrapulmonary accumulation of hemopoietic cells in nontransgenic, ovalbumin-sensitized recipients. a) intrapulmonary accumulation of hemopoietic cells is independent of local immunological injury induced by the allergen challenge; b) lung grafts systemically release IL-5, which is required for accumulation of hemopoietic cells in the recipientsT lungs; c) IL-5 is fully effective only on sensitized animals. Traditionally considered terminal effector cells, eosinophls are currently emerging in more subtle roles relating to their influence on tissue milieu and immunomodulation. This relatively new appreciation stemmed in part from the realizations that eosinophils store arsenals of preformed cytokines and chemokines with welldefined immunomodulatory properties, and are capable of rapid release of these potent mediators in response to specific stimuli. Although multiple mediators are stored in close proximity within eosinophil specific granules, their release appears to be independently regulated (i.e. To date, mechanisms governing this selectivity have been elusive. Interestingly, eosinophils are responsive to many of the factors for which they are reservoirs, indicating that cognate receptors for these ligands are also expressed. Despite the dual expression of receptor/ligand pairs by eosinophils, few studies have addressed receptor expression in relation to the storage and release of cognate ligands. In this study we utilize flow cytometric analysis to monitor intra-and extracellular expression of IL-4 and the IL-4 receptor. In an approach combining electron microscopy, light microscopy and subcellular fractionation, we further visualize localization of the IL-4 receptor throughout eotaxin-induced secretion of its ligand, IL-4. Surprisingly, we discover that in addition to nominal surface expression, all components of functional types I and II receptor complexes are pre-formed and stored within freshly isolated eosinophils. Further, we demonstrate the IL-4 binding component (IL-4 receptor a chain) is selectively mobilized in concert with IL-4 and likely participates in the trafficking of IL-4 out of the granule and through the vesicular compartment. This work represents the first indication of preformed internal stores of cytokine receptors within human eosinophils, and proposes a novel mechanism for the selectivity of mediator release.Our results demonstrate that SMILEYn is a brief, easily understood and valid pediatric SLE-specific QOL scale. Lack of significant correlation between child/parent reports suggests that we may be measuring different information from children/parents; childrenTs perception of their QOL may be different from their parentTs perceptions. [ A fractal analysis is used to model the binding and dissociation kinetics of biomedical analytes like connective tissue interstitial glucose, adipose tissue interstitial glucose, insulin and other related analytes. The analysis provides insights into diffusion-limited analytereceptor reactions occurring on heterogeneous biosensor surfaces.The fractal analysis is applied to the binding of glucose and other related analytes in solution to glucose derivatives immobilized on a biosensor chip. The fractal analysis provides a useful lumped parameter(s) analysis for the diffusion-limited reaction occurring on a heterogeneous surface via the fractal dimension and the rate coefficient. It is a convenient means to make the degree of heterogeneity that exists on the surface more quantitative. The fractal approach provides additional information about interactions that may not be obtained by a conventional analysis of biosensor data.Expressions of mIL-23 in murine colon carcinoma cells can induce murine macrophages to secrete higher levels of NO and TNF-a and has anti-tumor effect. Objective of the study: Peripheral T cell homeostasis is accomplished by balancing between proliferation and apoptosis. This is essential in establishing prompt immune responses but at the same time to avoid hypersensitivity reactions, immunemediated diseases, and lymphoproliferative disorders. Apoptosis is controlled via two different pathways. Antigen stimulation can induce Fas-dependent cell death, called activation-induced cell death (AICD), whereas absence of survival signals leads to activated T cell autonomous death (ACAD). Human adenotonsillar CD4+ T cells are critically located at the point of entry of foreign inhaled and digested antigens in the pharynx. This population includes cells that show expression of activation antigens. We used these in vivo activated adenotonsillar CD4+ T cells as a model to study homeostasis of peripheral CD4+ T cells. Materials and methods: Adenotonsillar tissue samples were obtained from children aged 1 to 4 years who underwent surgery because of adenotonsillar hyperplasia or infections. Naive phenotype CD4+ CD45RA+ and memory phenotype CD4+ CD45R0+ T cells were purified from homogenized tissues after ficoll centrifugation using antibodies conjugated with magnetic beads. To stimulate cells through TCR, cells were incubated with different concentrations of CD3 antibody. The involvement of the Fas-FasL interactions was determined using FasL antibody and recombinant Fas protein. To inhibit apoptosis, cells were treated with cytokines. Apoptosis was detected by analyzing phosphatidyl-serine translocation, DNA degradation, and caspase-3 activity. Results: Some adenotonsillar naive phenotype CD4+ CD45RA+ and most memory phenotype CD4+ CD45R0+ T cells expressed high levels of activation antigens, such as CD69. CD4+ CD45RA+ T cells, but not CD4+ CD45R0+ T cells, were sensitive to Fas-dependent apoptosis upon stimulation with a high concentration of anti-CD3 antibody. CD4+ CD45R0+ T cells were susceptible to rapid and spontaneous apoptosis in vitro that could be partially inhibited by cytokines IL-2, IL-15, and IL-7 that bind to the cytokine receptor common gamma chain, the chemokine CXCL12, and by IL-6, but not by interfering Fas-FasL engagement or TCR signaling, or by elimination of reactive oxygen species by a synthetic superoxide dismutase mimetic. Conclusions: Apoptosis of adenoidal naive phenotype CD4+ CD45RA+ T cells could be induced with a high concentration on anti-TCR antibody and was Fas-dependent, thus resembling AICD-type cell death. Apoptosis of CD4+ CD45R0+ T cells was reminiscent to ACAD-type cell death as it could be inhibited by various cytokines and it was not Fas or TCR dependent. Thereby, homeostasis of peripheral human CD4+ T cells is first achieved by deletion of naive CD45RA+ T cells by high concentrations of antigens, such as auto-antigens or nutrients. The magnitude of the immune response is then fine-tuned by various cytokines that can inhibit the death of activated memory phenotype CD45R0+ T cells.Our results revealed several differences in chronic patients when compared with subacute patients, including an increase in CD4 + /CD8 + ratio and an increase in Th2-like response. Background: The chemokine receptor CCR7 has been used in previous studies in combination with the CD45RA antigen as a tool to define different populations of memory CD4 and CD8 T lymphocytes with different homing and function capacities and at different stages of differentiation. Using this approach four populations of memory CD4 and CD8 T lymphocytes have been defined in patients with subacute and chronic hypersensitivity pneumonitis (HP) in bronchoalveolar lavage and blood. The following pattern of differentiation of CD4 and CD8 T cells has been demonstrated: CD45RA + CCR7 +Y CD45RA-CCR7 + Y CD45R AÀC CR7 À Y CD45RA + CCR 7À .Methods: Peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage cells (BALc) obtained from 5 subacute and 5 chronic HP patients were stained with mAbs to CD4, CD8, CCR7, CD45RA, CD62L. The secretion of IFN-g following specific stimulation was determined on antigen-specific CD4 and CD8 lymphocytes and this was used to identify specific CD4 and CD8 effector cells.Results: The CD8 memory blood T lymphocytes was equally composed pre-terminally differentiated CD45RA À CCR7 À (CD8 subacute: 30 F 12%; CD8 chronic 30 F 2.1 vs controls 10.5 F 2.3% P b 0.05) and terminally differentiated CD45RA + CCR7-(CD8 subacute: 20 F 7%; CD8 chronic: 30 F 14% vs controls 15.2 F 3.7) in subacute and chronic patients. In contrast CD4 blood T cells was composed pre-terminally differentiated (CD4 subacute: 34 F 15%; CD4 chronic: 38 F 27% vs controls 10.3 F 3.8 P b 0.05) and a higher proportion of terminally diferentiated memory CD4 T lymphocytes (CD4 subacute: 66 F 16%; CD4 chronic: 62 F 28% vs controls 16. 1 F 3.5 pb0.01 Background: Natural Killer cells (NK) play a pivotal role in the innate immune response, controlling tumour and infectionrelated responses. The cytolytic activity of NK cells is controlled by an array of activating and inhibitory cell surface receptors, including the killer immunoglobulin-like receptors (KIRs), which can be grouped into the most common haplotype groups called A and B. Cytokine changes occur in ageing and we wondered whether changes in serum cytokines were related to group A and B KIR haplotypes.Methods: KIR haplotyping available from 300 subjects from the Belfast Elderly Longitudinal Free-living Aging Study (BELFAST) was matched with a subset of corresponding serum cytokines.Results: Subjects categorized into the KIR haplotype associated with activation, showed increased serum cytokine values for both pro-inflammatory cytokines IL-2, IL-6, sIL-6, TNFa and IL-12 as well as increased values for the anti-inflammatory cytokines IL-10 and TNFb. There was no apparent sex-related difference.Conclusions: KIR haplotypes A and B in octo/nonagenarians do track with their activation status in relation to cytokines and could be effectors of tumour and infection-related responses and the immune-related changes in ageing.Since DC play a central role in T-cell activation, and in immunoglobulin synthesis, a failure of DC to mature into fully stimulatory cells and to present antigens may provide a more general explanation for the various symptoms of CVID patients. Multiple defects in the immune system, including malfunctioning of DC, appear to be prominent features of CVID patients. Objective: To describe the clinical and social demographic profile of patients with Primary Immunodeficiencies Diseases followed up at HC-UFMG. Methods: The patients followed up at HC-UFMG, diagnosed as Primary Immunodeficiency Disease according international consesus criteria, were studied. In these patients, a questionnaire was applied after informed consent, obtained from the parents and patients. It was performed nutritional evaluation by body mass index for age. The statistical analysis was performed using Epiinfo 6.0 package. The followed diseases were found: common variable immunodeficiency, X-linked agammaglobulinemia, C1-esterase deficiency, IgA-deficiency, IgG subclass deficiency, specific antibody deficiency, chronic granulomatous disease and ciclic neutropenia. They present also allergic, autoimmune and chronic inflammatory disease. The careful investigation primary immunodeficiency characterized by early onset of recurrent and severe infections. The molecular defects causing CGD are heterogeneous and lead to absence, low expression, or malfunctioning of one of the phagocyte NADPH oxidase components. The aim of this work was to analyze the clinical features and to investigate the molecular genetic defects of Latin American patients with CGD.Procedures: The study included 14 patients. The diagnosis was based on a history of recurrent severe infections, impaired respiratory burst, and the demonstration of an underlying mutation by single strand conformation polymorphism (SSCP) or RT-PCR analysis, followed by genomic DNA or cDNA sequencing.Results: Seven unrelated patients were found to have the Xlinked form of CGD. Heterogeneous mutations affected the CYBB gene: 2 insertions, 1 substitution, and 4 splice site defects; two of them are novel. Pneumonia was the most frequent clinical feature, followed by pyodermatitis, sinusitis, otitis, and liver abscess. Patients with X-CGD were more likely to have initial infections before age 2 years and to have inflammatory obstructive granulomas later. None of the patients had severe adverse reactions to BCG immunization.Conclusions: X-CGD patients from Latin America showed a high degree of molecular heterogeneity, including two novel mutations. Their clinical characteristics included early onset of infections and eventual obstructive granulomas. A47-CGD represented 50% of the reported cases, a higher prevalence than reported in other series. Hereditary angioedema is an uncommon condition which usually first presents in childhood. In the absence of appropriate treatment hereditary angioedema has a high mortality. There are a number of reports of successful short and long-term treatment with individual agents in those who suffer from hereditary angioedema, and the availability of these agents varies internationally. A recently published international consensus statement provides guidance for the management of such patients in adulthood, but there remains little guidance in the literature for the management of children and adolescents with hereditary angioedema. The needs of this age group are different to those of adults-in particular their symptomatology differs from adults, and the adverse effects of androgen treatment are different in children and adolescents. Here we review the treatment options available for children and adolescents with hereditary angioedema, and propose a simple evidence based guideline for the management of such patients.Su2.14. Abnormal NeutrophilTs Chemotactic Activity in Children with Congenital Insensitivity to Pain with Anhidrosis (CIPA): The Role of Nerve Growth Factor in Chemotactic Activity. Congenital insensitivity to pain with anhidrosis (CIPA) is a rare and severe genetic disorder. Loss of pain sensation leads to skin lacerations followed by deep tissue infections. CIPA has a relatively high prevalence in the consanguineous Israeli-Bedouin population, who carry a common mutation in the TrkA gene encoding the tyrosine kinase receptor for nerve growth factor (NGF). This defect in the NGF receptor results in complete absence of nonmyelinated and small myelinated fibers in the dorsal root ganglia. NGF has been shown to have chemotactic activity on neutrophils in-vitro and in animal models. We analyzed neutrophil functions in children diagnosed with CIPA and hypothesized that neutrophil chemotaxis is impaired as a result of the abnormal activity of the TrkA receptor.Twelve children genetically diagnosed with CIPA and carrying a 1926 ins-T mutation in the gene encoding for TrkA were recruited for the study. Two independent analyses of neutrophil functions including: chemotaxis, superoxide generation and phagocytosis, were performed in each child. Superoxide production was measured by reduction of acetyl-ferricytochrome c after stimulation of PMA, OZ and fMLP. Phagocytosis was assayed by ingestion of zymosan particles opsonized by pooled human serum.Chemotactic migration of CIPA patientsT neutrophils was significantly suppressed compared to controls (68.9 F 4.7%), while superoxide production and phagocytosis were normal. NGF alone did not act as a chemoatractant to neutrophils obtained from healthy donors. However, in the presence of NGF, neutrophils migration toward fMLP was elevated.Our results demonstrate an impaired chemotactic activity of neutrophils in CIPA patients. The chemotactic defect can account for the high prevalence of severe staphylococcal skin and bone infections of these children. Decreased chemotactic migration can be attributed to the molecular defect in the TrkA receptor as it was revealed that NGF has a strong adjuvant effect on fMLP-directed migration of neutrophils. The mAb 2F5 is one of the few cloned human antibodies with strong neutralizing activity against a broad range of HIV-1 laboratory and primary isolates. Using synthetic peptides containing the ELDKWA sequence from the gp41 membrane-proximal region recognized by the mAb 2F5 and mAb 2F5 as a positive control, we assayed in ELISA the reactivity with ELDKWA of IgG purified from sera of 30 Mexican patients with disease progression prepared prior to and after the initiation of Highly Active Antiretroviral Therapy (HAART). We found all sera to contain different but substantial levels of antibody reacting with ELDKWA. In addition, initiation of HAART led to elevation of the seroreactivity, which decreased gradually to the initial level after 24-hours of therapy. Serum of a patient with highest reactivity was investigated in a detail and shown the reactivity level to correlate with the decrease in viral load. The detection of 2F5-like antibody in all 30 patients, not reported for other countries, may either be explained by differences in the infection phases investigated or assigned to the local genetic variations of HIV-1 found in recent epidemiological survey. Since the epitope eliciting the antibody is conserved in clade B population, and the epitope is linear and available in form of synthetic peptide as antigen, we explore the possibility that the seroreactivity with ELDKWA peptides may be a diagnostic marker of the infection and disease progression in this country. Further studies are in progress to verify the hypothesis that the observed elevation of ELDKWA-seroreactivity by the therapy is due to the improved immunological status resulting from a suppression of the viral load in the critical phase of the disease progression. The innate immune defence is important for preventing infections or for eliminating microorganisms, which have penetrated the mechanical barriers of the body. Cell-bound as well as humoral molecules participate in recognizing the microorganisms by their pathogen-associated molecular patterns (PAMPs). Among the first group (termed pathogen recognizing receptors, PRRs) are the Toll-like proteins, while collectins and ficolins represent the latter group, and thus belong to the broader group of pathogen recognizing molecules, or PRMs. One of the collectins, mannan-binding lectin (MBL) and two ficolins, Hand L-ficolin are capable of activating the complement system. MBL, H-ficolin and L-ficolin each, thanks to their oligomeric structure provides for 12 or more clustered recognition domains, which allows for high avidity binding to suitably spaced PAMPs. MBL, and the mentioned ficolins are all three found in complex with proenzymes, named MBL-associated serine proteases (MASPs). After recognition of PAMPS the binding allows for the activation of the MBL-associated serine proteases. MASP-2 in turn activates C4 and C2 to generate the C3 convertase, C4bC2b. The outcome is the direct killing of the invader by the membrane attack complex or opsonization and killing via phagocytosis.When examining about 100 patients with suspect immunodeficiency we identified one patient who had normal levels of MBL but did not have the capacity to activate the complement system when probed on a carbohydrate surface. The patient was found to be homozygous for a SNP in the first domain of MASP-2 (the CUB1 domain) (Steengard-Petersen et al, New Eng J Med, 2003) . We have since observed homozygocity in two more patients. The gene frequency of 5.5 % stated in the above mentioned paper was based on 100 blood donors and was independently verified; however it appears to be a statistical aberration. We did not observe this MASP-2 SNP when analyzing samples from 200 healthy Asians from Hong Kong.As innate immune system factors may be involved in poor outcome in cystic fibrosis 112 of such patients (aged 4-45 years), were screened for MBL and MASP-2 levels and for MBL genotypes and for the MASP-2 SNP. We observed a higher frequency of the MASP-2 SNP in this patient population (no homozygous individuals) but no correlation to lung function was seen with MASP-2 SNP or with MASP-2 levels.Theoretically MASP-2 deficiency is a more serious condition than MBL deficiency, since MASP-2 is also required for complement activation by the H-and L-ficolin. Anaphylaxis to Intravenous and Oral Cyclosporin in a Child and Successful Desensitization. Hypersensitivity reactions to cylosporin are rare. Cyclosporin formulations for parental and oral use are vital drugs after bone marrow transplantation (BMT), thus recognition of hypersensitivity reactions and guidelines for subsequent use are important in transplant surveillance. The purpose of this paper is to report a case of anaphylaxis to intravenous and oral cyclosporin successfully managed by oral desensitization also to present a review of different formulations of cyclosporin with the least drug reaction. Case report: This 9-year-old girl with thalassemia major was admitted post BMT, when developed an anaphylactic reaction (respiratory distress, hypotension and generalized urticaria) after second exposure with intravenous then oral cyclosporin. Fortunately she had a good response to immediate rescue treatment. There is not any immunosuppressive drug as effective as cyclosporin for the engraftment. Two available formulations of cyclosporin in Iran do contain Cremophor-EL (polyoxyethylated caster oil) in IV or poly-5-oleate (a chemically similar compound to cremophor-EL) in oral compounds. The previous reported cases of cyclosporin hypersensitivity were confirmed to be due to this solubilizing agent rather than the cyclosporin itself. The safest suggested formulation, corn-oil-based soft gelatin capsule, was not available for us, thus oral desensitization was started according to the classic penicillin desensitization protocol and tolerated appropriately by the patient. There are a few reports of cyclosporin desensitization in the literature. Cyclosporine anaphylaxis is rare but possible, and in the face with unavailability of the suitable formulation, desensitization should be considered. Skin Reactivity to Aeroallergens Is Not Related to the Nasal Polyp Tissue Eosinophil Inflammation.Fardin Eghtedari, 1 Seyed Reza Cheraghzadeh, 2 Sara Kashef, 3 Ahmad Monabati, 4 Elham Shoraka. 5 The role of allergy in the pathogenesis of nasal polyposis is not clear. In this study we investigated the possible correlation of skin reactivity to aeroallergens, with the polyp tissue eosinophil inflammation. Twenty-five patients with nasal polyposis who were candidate for polypectomy under general anesthesia were enrolled. Polyp tissues were stained with hematoxilin-eosin for eosinophil count. Skin prick test (SPT) with at least 11 common aeroallergens (Allergopharma, Germany) including pollens, mites and molds were done for all patients. The positive SPT was defined as a reaction at least 3mm larger than the negative control (Glycerol). In 18 patients eosinophil count in the polyp tissue was more than 50 percent of cells counted in the field. We did not find a significant correlation between the polyp eosinophil count comparing to the skin reactivity. It seems that polyp eosinophil inflammation is not a consequence of allergy to the aeroallergens in the nasal polyposis. Allergen immunotherapy is the only treatment for allergic rhinitis and asthma that can reverse the immune imbalance in patients with these IgE-mediated disorders. This form of therapy involves gradual administration of increasing doses of allergens to patients who have been found to possess allergen-specific IgE reactivity. The treatment is 90% effective in reducing both allergy symptoms and medication use, while improving the quality of life for the allergy sufferer. Allergen immunotherapy with aqueous allergens, however, carries the risk of systemic reactions. Using a questionnaire of its membership, the Immunotherapy Committee of the American Academy of Allergy, Asthma and Immunology verified the potential risks of both death and near death reactions immediately following administration of aqueous allergens. Their findings confirmed that there had been 273 near death reactions and 20 deaths associated with aqueous immunotherapy from 1990 to 2001.To reduce life-threatening reactions to allergen immunotherapy, Patterson developed the technique of glutaraldehyde polymerization of ragweed and grass allergens. In multiple studies, polymerized vaccines were found to be as effective as aqueous allergen extracts, and devoid of systemic responses. Here we extend PattersonTs findings and demonstrate that polymerized ragweed and grass vaccines are superior in safety to aqueous materials. 500 allergy patients were given over 55,000 injections of polymerized ragweed and grass allergens, with zero systemic responses.To eliminate the risk of death associated with aqueous allergen immunotherapy, while retaining its effectiveness, we strongly recommend that allergy patients receive polymerized ragweed and grass vaccines. Given that the use of aqueous allergen immunotherapy by untrained physicians and nurse practitioners is increasing nationwide, it may be safer for patients to have the FDA either eliminate the availability of aqueous ragweed and grass allergens altogether, or restrict their use to physicians board certified in Allergy-Immunology. L. Chini, 1 F. Angelini, 1 C. Chatgilialoglu, 2 S. Dellonte, 2 V. Moschese, 1 S. Corrente, 1 R. Iannini, 1 M. Chianca, 1 P. Rossi, 1 C. Ferreri. 2 1 Pediatrics, Policlinico Tor Vergata, University of Rome Tor Vergata, Rome, Italy; 2 ISOF, Consiglio Nazionale delle Ricerche, Bologna, Italy.The formation of trans fatty acid residues in membrane lipids can be due to the radical-catalysed isomerization process of naturally occurring cis fatty acid moieties. Radical stress is well documented in atopic diseases but no data are still available on a possible association with high levels of trans fatty acids in these patients. We investigated the presence of trans lipid isomers in erythrocyte and T-lymphocyte membranes of 24 children affected by atopic eczema/dermatitis syndrome (AEDS) taking advantage of the trans lipid library available from radical processes modelled in vitro. We found trans fatty acids both in erythrocyte and lymphocyte membranes and their total content reached the highest value of 3.0% of the main fatty acid residues. Moreover, the highest trans fatty acid levels were detected in 12 out of 24 children which have atopic dermatitis not mediated by IgE (prick/RAST negative). A new significance of lipid impairment in AEDS can be proposed, which generally involves the role of trans isomers in human pathologies. This study aims to contributing to lipidomic researches regarding the double bond structure and the influence of a geometrical change of membrane lipids in physiology and diseases. Proinflammatory Cytokines and Nitric Oxide in Exhaled Breth Condensate in Monitoring of Exacerbation Asthma in Children. Boleslaw Kalicki, 1 Anna Jung, 1 Wanda Stankiewicz, 2 Marek Dabrowski, 2 Janusz Zuber. 1 1 Paediatric, Military Medical Institute, Warsaw, Poland; 2 Immunology, Institute of Hygiene and Epidemiology, Warsaw, Poland.Proinflammatory cytokines and nitrici oxiden play important role in exacerbation of asthma. The aim of the study was to determine NO, IL-4, IL-6 level in exhaled breath condesate of asthmatic children.Material and methods. The samples of exhaled breath condensate were collected in 31 children with asthma (16 females and 15 males, aged 8-18y, mean 13,3y) during 15min. breathing and then were frozen to (-) 70 8C. The examination of the exhaled breath condensate were done by EcoScreen equipment (Jaeger Comp.). Results were compared in 3 group of asthmatic children (I group-7 children with asthma exacerbation, II group-9 children without exacerbation of asthma, well controlled by steroids and h2mimetic drugs, III group-15 children with asthma improvement without drugs longer than 3 months) and control (IV-th group-15 healthy children aged 11-17y, mean 14,5y).Results. The highest value of NO, IL-6, IL-4 were found in I group of children. All parameters have shown significant differences between examination groups.Conclusions. Mean concentrations of NO and cytokines IL-6, IL-4 had strong correlation with exacerbation asthma in children.2. The examinations of NO in exhaled breath condensate especially but also IL-6, IL-4 cytokines are useful, non-invasive method in monitoring exacerbation asthma in children. Introduction: In children with probable peanut allergy, an 8-mm skin prick test (SPT) has been reported to be 100% specific. We aimed to determine the sensitivity and specificity, for peanut allergy, of skin tests, peanut specific-IgE, and combinations of these, in children with a lower pre-existing probability of peanut allergy. Methods: Children attending the allergy clinic with a positive peanut SPT (n = 84; age range 0.9-17.3 years; mean 4.5 years) were included in the study. Immediate skin application food tests (I-SAFT) using 1 gram of peanut butter (positive if any wheals were detected at 15 minutes), peanut specific-IgE levels and open-label peanut food challenges were performed. Results: Fifty-two of 85 peanut challenges were positive. Combinations of an 8-mm SPT with a positive I-SAFT and a peanut specific-IgE N 0.35 kU/L were 88% specific. Conclusion: An 8-mm SPT cannot predict peanut allergy in children without a high pre-existing probability of peanut allergy. If a child without a recent history of a peanut reaction has a SPT of b 15-mm diameter, peanut specific-IgE should be measured. Allergy test results should be interpreted in the context of a history or suspicion of food allergy. CowTs milk allergy has been considered as a cause of infantile colic. In this study, we evaluated the role of cowTs milk allergy in infantile colic in a group of exclusively breast fed infants. 114 exclusively breast fed infants between three weeks and three months of age, who were referred with infantile colic, enrolled in this study. Skin prick test with cowTs milk extract (Allergopharma) and a stool exam for occult blood were done for all babies. Then, they were randomly selected as two groups of case and control. In case group (including two babies with positive skin prick test), we advised mothers not to consume cowTs milk and other dairy products for two weeks. In control group, we did not change the diet of mothers. Infants with colic whose mothers did not take dairy products, did not improve significantly in comparison with control group. Prevalence of positive skin prick test in colicky infants was 2.6 %, which is nearly similar to prevalence of cowTs milk allergy in the population of infants below one year of age (2.2-2.6% on the basis of previous studies). Occult bleeding in stool was significantly higher in colicky infants in comparison with non-colicky infants. CowTs milk allergy does not seem to be a common cause of infantile colic. It is not advised to eliminate the dairy products from the diet of nursing mother. 5-LO in different cells may variably be present in the cytosol and/or the nucleus and may undergo activation-dependent translocation to sites, including the nuclear envelope. Lipid bodies are organelles that in leukocytes and other cells have roles in the local formation of both 5-LO-and cyclooxygenase pathway-derived eicosanoids. We have evaluated the expression of 5-LO in rat basophil leukemia cells (RBL-2H3). Resting RBL cells contained numerous lipid bodies, as identified by staining with Oil Red O and the incorporation of a fluorescent fatty acid analog. By immunocytochemistry, 5-LO was present in the cytosol and nucleus of resting RBL cells, as well as at punctate cytosolic sites, that costained as lipid bodies. Resting RBL cells were disrupted by nitrogen cavitation and subjected to subcellular fractionation with a protocol designed to isolate buoyant lipid bodies. By Western blotting of subcellular fractions, 5-LO was present in lipid body as well as cytosolic and nuclear fractions. To investigate the localization of 5-LO within RBL cells, cells were transfected a plasmid encoding an EGFP-5-LO fusion protein. Examination of cells as soon as 1 hr after transfection with EGFP-5-LO demonstrated very prominent focal green fluorescence at punctate cytosolic sites that stained as lipid bodies with Oil Red O. EGFP-5-LO fluorescence remained largely lipid body associated at 4 hrs post-transfection, when a lesser number of cells also began to exhibit diffuse cytosolic fluorescence. To ascertain whether cell activation altered the EGFP-5-LO distribution, cells were sensitized with anti-DNP IgE and activated with DNP. At both 1 and 4 hrs after IgE-mediated activation, lipid body numbers per cell increased~50%. At 1 and 4 hrs after activation, EGFP-5-LO fluorescence exhibited almost exclusively punctate cytosolic localization with lipid bodies. Thus, lipid bodies in RBL cells constitute a discrete pool of 5-LO that is especially enriched in newly synthesized 5-LO. These findings provide additional evidence for the functions of lipid body organelles in the formation of eicosanoids pertinent to inflammation.Category Potentially serious immune reactions or loss of treatment effect may result from repeated exposure of patients to therapeutic proteins or peptides even when they are from human sources or based on human sequences. Materials and Methods: The package inserts of twenty therapeutic human protein/peptide products were examined for the following information: alterations to peptide sequence or polysaccharide attachment, manufacturing process, choice of excipients, viral validation, assay of antibody development, in vitro or in vivo correlates of cell mediated immunity, precautions and contraindications in specific patient populations, therapeutic effect, and adverse reactions. The products included hormones, cytokines, coagulation factors, immune globulins, as well as other blood components. Results: (1) Information on changes potentially affecting immunogenicity, such as differences in conforma-tion of the therapeutic agent molecule and the possible adjuvant effect of certain excipients, is rarely presented except for some recombinant products. (2) Immunogenicity is most often studied for the development of serum antibodies to the therapeutic agent, but rarely in terms of cell-mediated immune responses or skinsensitizing antibodies, unless the route of administration is dermal application. (3) Instructions on testing for antibodies to the therapeutic agent are often dictated by changes in treatment effect, and only occasionally by manifestations of adverse reactions, such as anaphylaxis. (4) Special populations, which are susceptible to the development of immune responses to the therapeutic agent, are generally addressed in the precautions, warnings and contraindications sections of labeling. However, systematic studies to explore immunogenicity in specific populations are often lacking. Conclusions: Information regarding immune reactions to human proteins/peptides remains inadequately pursued or presented for many available therapeutic products. Greater attention needs to be focused on this important issue, which has bearing on both safety and effectiveness of the products. Soluble IL-4 receptor (sIL-4r), functioning as a decoy receptor, inhibits action of IL-4. The aim of this study was to evaluate serum concentration of sIL-4r in asthma patients during bronchial challenge with Dp allergen.The study was performed on 51 asthma patients with a positive history of dust allergy symptoms, positive skin prick test results with Dp extract and with a significant bronchoconstrictive response to bronchial Dp challenge. Ten healthy persons with negative skin prick tests to common aeroallergens were used as controls. Bronchial provocation challenge with Dp extract was performed only in asthma patients. Blood samples were collected before, 1 hour (T EAR ) after, 8 hours (T LAR ) after and 24 hours (T 24 ) after allergen challenge. Plasma concentration of sIL-4r was evaluated by ELISA (R&D Systems).The mean plasma concentration of sIL-4r was greater in asthma patients (46.6 F 18.6 pg/ml) than in healthy controls (29.1 F 14.5 pg/ml). There was no difference in the mean plasma concentration of sIL-4r between patients who responded to allergen challenge with isolated early asthmatic response (single responders-SR) and those who responded with both early and late asthmatic responses (dual responders-DR). During the T EAR , a significant fall in plasma sIL-4r concentration was found in DR (to 33.7 F 12.6 pg/ml; P b 0.001), but not in SR (45.8 F 23.6 pg/ml). At T 24 the mean plasma concentration of sIL-4r was significantly greater in SR (57.9 F 27.9 pg/ml) than in DR (41.1 F 13.4 pg/ml), but was not significantly different from the baseline levels.The fall in sIL-4r plasma concentration in DR seen at T EAR may result in increased activity of IL-4 which in turn may participate in the development of sustained allergic inflammation in these patients. The interleukin-4 (IL-4) splice variant (IL-4y2) is known to antagonize many biological activities of IL-4. The aim of the study was to compare the IL-4 and IL-4y2 expression ratio in patients with asthma versus healthy subjects.Materials and methods. Eight healthy subjects and eight patients with atopic asthma confirmed by case history, skin tests and RAST were involved in the study. RNA was extracted from PBMC obtained by Ficoll-Urographin gradient centrifugation. cDNA was used for quantitative PCR with specific form-discriminating primers and SYBR Green I performed using iCycler iQ (BioRad, Hercules, USA).Results. The IL-4:IL-4y2 ratio did not correlate with age, sex, total serum IgE levels, or the presence of eczema, rhinitis or anaphylaxis in the patients with asthma.Conclusion. There was no difference in relative expression of splice variant of IL-4 in healthy subjects versus atopic asthma patients. HIL-4y2 is a natural alternative splicing form of human IL-4, derived from hIL-4 mRNA by the elimination of second exone encoding for the amino acid residues 22-37. The structural and biological properties of IL-4y2 are poorly understood, and its direct (non-mRNA) quantitative measurement is currently not possible. The major purpose of this study was: a) to compare the 3D structures of hIL-4y2 to the parent cytokine hIL-4; b) to attempt to reveal the dominant B epitope sequences in loop regions of hIL-4y2 using synthetic peptides and computer-assistant 3D modeling; c) to prepare monoclonal antibodies (MAbs) specific for each cytokine form; and d) to evaluate the binding affinities of the antibodies. The hIL-4/hIL-4y2 peptides were synthesized by solid-phase methods, characterized by analytical HPLC and mass-spectrometry. To increase immunogenicity, the peptides were conjugated to keyhole limpet hemocyanine (KLH) and used as immunogens. Polyclonal and monoclonal antibodies to both hIL-4 and hIL-4y2 peptides in addition to whole recombinant proteins were produced in BALB/c mice. Analysis of reactivity of mouse antisera produced against hIL-4 and hIL-4y2 showed very low reactivity to all synthetic peptides, while anti-peptide antisera obtained demonstrated noticeable reactivity to IL-4, especially the anti-serum to the peptide mimicked splicing fragment consisting of sequence 22-37 of IL-4. ImmunoDot and ELISA demonstrated that anti-IL-4 MAbs produced were able to recognize only peptide 22-37. Reactivity of anti-IL-4 peptide antisera to hIL-4y2 was completely absent. A 3D model of IL-4y2 which was used as a template to design the peptide mimicked the unique B epitope which emerged in the splicing site. Assay of antibody binding of MAbs to I 125 labeled cytokines showed that K50 values varied from 10 À10 to 10 À6 M. These techniques may allow for identification and further characterization of differences between hIL-4y2 and IL-4, including both quantitative and qualitative functional aspects. Endotoxin has been shown to have a powerful effect on adaptive immunity. Although endotoxin, in particular, LPS is an important adjuvant during the priming of allergic immune responses, it has been shown to suppress recall Th2 immune responses. We tested the effect of LPS on in vivo priming and memory responses in a model of allergic asthma induced without adjuvants. Mice were immunized twice on days 0 and 21 with 10 mcg of ovalbumin (OVA) intraperitoneally. One week later, mice were challenged with a series of 4 aerosolizations with 1% OVA on 2 consecutive days. Groups of mice were either evaluated for acute disease or recuperated for at least 2 months before being rechallenged for relapse disease. We found that mice sensitized intraperitoneally with OVA with very low LPS content (removed with detoxigel; contained 50 pg/ml endotoxin) and rechallenged with OVA (Sigma, grade V; contained 190 ng/ml endotoxin), developed minimal allergic lung inflammation, mucus secretion, and OVA-specific IgE and IgG1, compared with animals immunized with high LPS containing OVA. However, when mice were immunized using the same protocol with low-fat milk powder (containing endotoxin 1.3 ng/ml) and aerosolized with a 1% milk solution, responses were significantly higher than low endotoxin containing OVA primed mice. These data indicate that LPS may be critical for inducing Th2 responses to OVA but is not necessary for priming to milk proteins. To test the effect of LPS on in vivo memory responses that lead to disease relapse, we challenged recuperated mice with OVA and titrated doses of LPS (1.0, 100.0, 1000.0 ng/ml) in the aerosol OVA solution. We found that there was inhibition of allergic inflammation, mucus production, and immunoglobulin at 1.0 and 1000.0 ng/ml added LPS but not at 100.0 ng/ml. To determine whether the LPS suppression on memory responses with low and high LPS doses involved B cells, we did the same experiment in B cell deficient mice. Interestingly, in the absence of B cells, we found suppression with intermediate LPS doses but no effect with high and low doses suggesting that B cells play a role in the LPS effect on recall memory responses. In summary, our results show that LPS is necessary during priming with certain proteins, such as OVA, but not with others, such as with milk. Additionally, LPS has an inhibitory effect on memory responses that appears to involve B cells. CowTs milk allergy is a significant health problem during infancy and childhood. Patients are usually allergic to all the major milk proteins in cowTs milk or formulas, including caseins, betalactoglobulin, and alpha-lactalbumin and as a result develop dermatitis, asthma, or anaphylaxis. Currently, there are no animal models of milk-induced allergic asthma. The advantages of milk as an allergen in experimental models is that it is natural and clinically relevant, broadens the scope and general applicability of mouse models of allergic asthma, induces an allergic response to a combination of proteins, and is cost effective (3 kg of milk powder in Vienna is 15.00 EUR; 50 g ovalbumin 900.00 EUR). To establish models of acute and relapse milk-induced allergic asthma in mice, we injected either BALB/c or C57BL/6 mice with 10 mcg of low-fat milk powder dissolved in PBS intraperitoneally three weeks apart. One week later we exposed mice to a series of 4 milk-aerosol challenges with a 2% solution of milk powder in PBS, on two consecutive days. Mice were either evaluated for disease during acute onset of disease or recuperated in the following 2 months and were then re-exposed to milk with a similar series of aerosol challenges to generate disease relapse. We observed increased lung inflammation, mucus secretion, and milk-specific IgE and IgG1 at acute onset disease (day 31) and during relapses (day 90) in both C57BL/6 and BALB/c mice. However, milk sensitization and aerosolization induced 10-fold higher infiltrating inflammatory cells in bronchoalveolar lavage fluid in C57BL/6 compared with BALB/c mice. The percent eosinophils in the airways were 70 F 0.7 % for C57BL/6 and 31 F 3.5% for BALB/c mice, compared to no eosinophils in naive and recovered mice. Furthermore, the number of perivascular and peribronchial infiltrates and eosinophils within the lungs in tissue sections reflected these differences. Similarly, mucus hypersecretion and milk-specific IgE and IgG1 levels were higher in C57BL/6 compared to BALB/c mice and all observed differences between strains were apparent in acute and relapse disease. Recuperated C57BL/6 and BALB/c mice had lymphocytic infiltrates in the lungs, as previously demonstrated in ovalbumin-induced models. In contrast to ovalbumin-induced allergic asthma, milk sensitization and aerosolization resulted in a large difference between mouse strains, which is not yet fully understood. Here, we present a useful, inexpensive, and clinically relevant mouse model of milk allergy in C57BL/6 mice and demonstrate significant genetic differences in immune responses of C57BL/6 and BALB/c to cowTs milk. Background: Asthma in older adults is under-recognized and is often associated with allergic triggers. Anti-immunoglobulin E (IgE) therapy with omalizumab (OMA) is indicated in patients (z 12 years) with moderate to severe allergic asthma who continue to be inadequately controlled despite treatment with inhaled corticosteroids. Previous analyses have not focused specifically on efficacy in older adults. We examined treatment response to OMA on asthma exacerbations as well as patient-reported and investigator-reported global treatment effectiveness in patients 50 years and older.Methods: Data were combined from 5 randomized double blind placebo-controlled (PBO) trials of patients with moderate to severe allergic asthma (confirmed by skin test or RAST testing); 4 were of 28 weeks and 1 was 32 weeks in duration. The pooled study population involved a total of 2236 patients (1136 treated with OMA, 1100 treated with PBO) who met entry criteria that included, at baseline, need for treatment with moderate to high dose inhaled corticosteroids. 601 subjects were z50 years of age. The relative risk (RR) of clinically significant asthma exacerbations (OMA vs. PBO; primary endpoint) was determined using Poisson regression, controlling for age category, study, sex, baseline IgE, and prior history of asthma exacerbations for the overall population and for patients z50 years. A similar approach was taken for evaluation of patient-and investigator-reported global treatment effectiveness (excellent, good, moderate, poor, worsening) using cumulative logistic regression for the two groups comparing OMA to PBO.Results: The mean age of the older subgroup was 58 years; 61% were female; the mean IgE level was 184 IU/dl (range 19-743). For the overall study population the mean age was 40 years (range 12-79); 58% female; the mean IgE level was 211 IU/dl (range 19-1055). OMA was associated with a reduced risk of clinically significant asthma exacerbations in all 5 trials reviewed. Pooled analysis in the overall study population revealed a RR (OMA vs. PBO) of 0.79 (95% CI 0.62-0.97). In the subgroup of patients 50 or older, the RR was 0.72 (95% CI 0.48-1.09). The improvement shown with OMA was in agreement with patient-and investigator-reported global effectiveness which demonstrated significantly greater response (P b 0.0001) on both measures in patients assigned to OMA relative to PBO irrespective of age.Conclusions: In patients z50 years of age, omalizumab was associated with a RR reduction in clinically significant asthma exacerbations and significantly better patient-and investigatorreported global effectiveness ratings compared to placebo, suggesting that omalizumab is effective in older patients with moderate to severe allergic asthma. Treatments were generally well tolerated. Our laboratory has demonstrated that human B lymphocytes synthesize IL-13, and that autocrine production of this cytokine appears to be essential for maintaining IgE production by these cells. To initiate IgE synthesis, contact between CD40 on B cells and CD40 ligand (CD40L or CD154) on Th2 cells is necessary. A culture system, using murine CD154-transfected fibroblasts (LTK) has been established for the propagation of human B cells in vitro. Our objective is to develop a model of IL-13-producing B cell using this co-culture system.Methods: Human B lymphocytes were isolated from tonsils and purified by sheep red blood cell rosetting. LTK cells were stably transfected with a cDNA encoding for the wild-type form of the human CD154 protein. In the co-culture system, 5 Â10 5 B cells were co-incubated with 1.3 Â 10 5 untransfected LTK (CD154-) cells or transfected LTK-4A1 (CD154+) cells and seeded in 24well plates. Isolated B cells were also stimulated with soluble anti-CD40 antibody (1Ag/ml). After 5 days of co-culture, detection of intracellular IL-13 in B cells was assessed by flow cytometry. We also measured levels of phosphorylated STAT6 by flow cytometry.Resultats: Using BrdU staining, we demonstrated that LTK-4A1 most efficiently supported survival of B lymphocytes with an increase in proliferation (41.36% versus to 9.59%) compared to soluble anti-CD40 antibodies. LTK-4A1 blocked apoptosis of B lymphocytes more efficiently than soluble anti-CD40 (39.7% vs. 13.77%). Most importantly, after 5 days in culture with LTK-4A1, the number of CD19+/IL-13+ B cells was significantly higher (47.9% versus 17.1%) compared to the soluble anti-CD40 Ab. The production of IgE by human B lymphocytes cultured with LTK-4A1, as assessed by ELISA, also increased significantly (13.4 ng/ ml versus to 1.8 ng/ml) when compared to B cells stimulated with soluble anti-CD40 antibodies. In contrast, IL-13 receptor signaling was equally influenced by both culture systems. STAT6 phosphorylation in response to exogenous IL-13 was nearly equal 24 hours after co-culture with LTK-4A1 or with soluble anti-CD40 compared to the untransfected LTK control (75% of phospho-STAT6 positive cells in co-culture with LTK-4A1 or with soluble anti-CD40 versus 10% with LTK).Conclusion: LTK-4A1 induces more efficient cross linking of CD40 than soluble CD40 antibodies. This leads, in turn to high levels of IL-13 positive B cells, not previously demonstrated. This likely suggests that IL-13 production by B cells is important in vivo. Th2 cytokine production by human B lymphocytes may be underreported due to incomplete stimulation via CD40. Rationale Daclizumab (ZenapaxR), a humanized monoclonal antibody directed against the IL-2 receptor a chain (CD25), is approved for the prevention of renal allograft rejection and is under evaluation for treatment of asthma, multiple sclerosis and other autoimmune diseases. Daclizumab inhibits activation of human T lymphocytes by blocking IL2-induced T cell proliferation, and by reducing production of Th2-and Th1-associated cytokines.Naturally occurring regulatory T cells (T Regs) are thought to play an important role in the prevention of autoimmune diseases in man and mouse (Sakaguchi, S. Annu Rev Immunol 22, 531-562 (2004) ). Since T Regs are characterized by the constitutive expression of high levels of CD25, we evaluated the in vitro effect of daclizumab on the function of these cells.Methods CD4 + T cells were enriched from whole blood obtained from healthy human donors using StemCell Technologies RosetteSepk system. T Regs were flow sorted as CD4 + CD25 bright (top 1.5% of CD25 staining intensity) and effector T cells (T Eff) were sorted as CD4 + CD25-(bottom 5% of CD25 staining intensity). The inhibitory activity of T Regs was assessed in a standard co-culture system. Briefly, 2Â10 4 T Eff alone, T Reg alone, and T Eff + T Reg were stimulated for 3 days in the presence of immobilized anti-CD3 in the presence of irradiated autologous APCs. Proliferation was measured by 3 H-thymidine incorporation during the last 16 hours of culture.Results T Regs stimulated for 3 days in the presence of daclizumab (10 Ag/mL) showed no or little proliferation, similar to T Regs stimulated in the absence of DAC. T Effs stimulated alone demonstrated substantial proliferation that was inhibited by daclizumab, on average by 40%. As expected, T Regs suppressed the proliferation of T Effs in the co-culture system. In this system, preincubation of T Regs with daclizumab did not affect the suppressive activity of these cells. Conclusions In these co-culture experiments, daclizumab had no effect on the function of T Reg cells but did inhibit T Eff cells from healthy human volunteers. Respiratory exposure to environmental antigens such as OVA induces rapid expansion of antigen specific T helper cell population in mice. Primary intranasal challenge with OVA also induces differentiation of activated OVA specific effector Th cells from naRve precursors. Effector function has been demonstrated in short-term culture with antigen re-stimulation. The effector Th cells are found both in draining lymph node and in circulation. However, secondary intranasal challenge with OVA fails to induce inflammation in the lung, even when the size of the Th cell population is at its peak. Based on these observations, Th cells primed in the respiratory system by environmental antigens in the absence of adjuvant are viewed as defective or anergic. In this study, we demonstrated the expression of effector function of intranasal primed Th cells in the lung following a challenge with antigen-bead emboli in C57BL6 mice. Importantly, secondary intranasal challenge with soluble antigen did not induce Th cell mediated inflammation in the lung. These results suggest that Th cells primed via respiratory route by environmental antigens are not anergic. More importantly, it is clear that the expression of Th cell effector function is tightly controlled by innate response in the lung. Bead emboli, but not soluble OVA, induced rapid increase of chemokine expression and rapid increase of the number of activated dendritic cells in the lung. However, it is not clear which innate events are critical for the expression of Th cell effector function in the lung. We propose that loss of innate regulation of Th cell effector function in a peripheral organ is necessary for T cell mediated organ specific disorders. Extracellular adenosine 5V-triphosphate (ATP) is a local physiologic regulator. We have previously shown that ATP stimulates bronchopulmonary vagal sensory terminals of nociceptive C and stretch-sensitive A fibers, and that this action is mediated by P2X receptors (R) (J Physiol (Lond) 490:265-75, 1996 , ditto 551:869-79, 2003 . The stimulatory action of ATP on C and A fibers could be involved in ATP-induced bronchoconstriction and cough. Vagal sensory neurons in the nodose ganglion express homomeric P2X2R and P2X3R as well as heteromeric P2X2/3R. To further explore the P2XR subtype that mediates ATPinduced action potentials (AP) in nodose C and A fiber terminals in the lungs, the effects of A-317491, a potent and selective antagonist at P2X3R and P2X2/3R sites (Proc Natl Acad Sci USA 99:17179-84; 2002) , on the activation of guinea-pig intrapulmonary vagal sensory nerve terminals by a,h-methylene-ATP (a,h mATP), a potent selective agonist at P2X3R and P2X2/3R sites, were studied in a perfused isolated lung preparation. The AP in C (n = 4) and A fibers (n = 7) induced by a,h mATP (10 AM, 1ml, bolus) in the absence and presence of A-317491 (1 and 10 uM, 30 min) were quantified as discharge/sec; data are mean + SD. a,h mATP induced AP in a non-desensitizing manner in both C and A fibers, the frequency was 146 F 29 and 1543 F 285, respectively. A-317491 (10 AM) reduced this response by 62 F 5% and 88 F 5%, respectively (P b .05). At 1 uM, A-317491 significantly inhibited the action of a,h mATP in A fibers by 59 F 12%, but had no inhibitory effect on C fibers. Conclusion: a,h mATP stimulates both nociceptive C and stretch-sensitive A fibers by acting on P2X2/3R. The present data could also indicate some difference in the nature of the P2XR subtype expressed on the two fiber phenotypes. In addition, since aerosolized ATP induces bronchoconstriction and cough in human subjects and more so in patients with asthma and COPD, A-31749 could constitute a novel therapeutic modality in the management of patients with chronic obstructive airway diseases. Support: Duska Therapeutics, Inc., Bala Cynwyd, Pennsylvania. The cell physiological basis for this is unknown. We have investigated the biological basis for sulfite sensitivity in a mast cell line (RBL-2H3), human peripheral blood basophils, and airway epithelial cells. RBL-2H3 cells were exposed to varying concentrations of sodium sulfite in the presence and absence of anti-oxidants and inhibitors of redox pathways. Sodium sulfite induced mast cell and basophil degranulation to a level equivalent to that induced by IgE cross-linking and ionomycin. The response was independent of extracellular calcium influx. Using a redox sensitive fluorescent dye, 2V7V-dichlorofluorescein diacetate, sulfite was shown to increase the generation of intracellular reactive oxygen species (ROS). Upregulation of sulfite-induced ROS generation was also demonstrated in the airway epithelial cell line, A549. Both ROS and degranulation induced by sulfite was inhibited by the free radical scavenger tetramethylthiourea and the flavoenzyme inhibitor diphenyleneiodinium. Overall, the data suggest that one potential mechanism of sulfite-induced asthmatic symptoms may be due to activation of airway mast cells and epithelial cells through the generation of ROS via activation of the NADPH oxidase complex with increased generation of superoxide anion. Cedar pollen hypersensitivity is a major cause of seasonal airway symptoms in several regions of the Northern Hemisphere. Group 1 allergens have been isolated, cloned and sequenced from the pollens of at least six cedar species. Given the high degree of amino acid sequence identity and evidence for immunologic cross-reactivity between cedar allergens, we investigated the structural basis for sharing of IgE epitopes between two group 1 allergens. Crossreactivity between Jun a 1 and Cry j 1 from Japanese cedar (JC) was probed with sera from JC-allergic patients by ImmunoCAP inhibition. Linear IgE epitopes were identified for Cry j 1 with an array of overlapping Jun a 1 peptides, using sera from patients allergic to JC. The binding of mouse monoclonal anti-Cry j 1 antibodies to these peptides were also tested. ImmunoCAP inhibition indicated that about 1/3 of the IgE anti-JC pollen antibodies in the sera of JC-allergic patients reacted with Jun a 1. One epitope, which maps to the betahelical core of Jun a 1 was not recognized by the sera of JC patients, despite complete sequence identity and apparent similarity of surface exposure. Monoclonal antibodies to Cry j 1 identified another shared epitope that is probably conformational and one unique Cry j 1 epitope, which may be a glycopeptide structure. These findings indicate that the IgE antibodies to several linear and conformational IgE epitopes of group 1 cedar allergens cross-react with homologous allergens. The shared responses may recognize structural elements common to many plant allergens. These findings suggest that patients from genetically diverse population respond to similar linear and conformational epitopes of homologous allergens. Understanding the similarity and difference in the immune responses to groups of allergens will aid in the development of more effective allergy vaccines. allergy admissions with admissions related to other acute allergic diseases.Methods: A database of all acute hospitalizations in New York state was examined from 1994-2003. The Statewide Planning and Cooperative Research System (SPARCS) database compiles mandatory reporting from acute care hospitals with an information input regarding diagnoses, disposition, procedures, insurance, demographics, and charges. Patient admissions with diagnosis (principal or otherwise) for food allergy (using ICD-9 codes V15.0, 995.6) or other allergic diseases including anaphylaxis, urticaria and allergy unspecified (ICD-9 codes of 995. 0, 995.1, 999.4, and 708) were extracted. Demographic characteristics were tabulated for the hospitalizations and patterns were examined. Admissions for food allergy were compared to admissions for other acute allergic diseases.Results: Over the decade examined, admissions for New York state hospitals which coded for allergic disease exclusive of food related codes increased only by less than 5%. However, the number of admissions for allergic conditions involving food allergy tripled. The median age for allergic disease related admissions increased over the years studied; however, the median age for food allergy admissions actually decreased. The increase in age for non-food related allergic disease admissions appeared to be primarily due to an increase in angioedema admissions where the age was greater. Food allergy related admissions increased more in non-African American patients than in African American patients over the decade study. In contrast, allergy related admissions exclusive of food related codes increased in African Americans, especially angioedema. Age related differences were observed with respect to specific foods causing anaphylaxis.Conclusions: Food allergy related hospitalizations are being increasingly reported in New York State. Whether an increase in food allergy related hospitalizations relate to and increase in food allergy or a greater awareness of these conditions cannot be determined, but further research on patterns of food allergy related hospitalization is clearly warranted. Antioxidant therapy might be a better strategy to augment endogenous antioxidants for better management of asthma. Vitamin E is a strong lipophilic antioxidant with multiple actions both at biochemical and cellular level. Effects of its supplementation with standard therapy have not been explored in asthmatics. We conducted a double blind standard therapy-controlled study to assess the role of exogenous supplementation of vitamin E on endogenous oxidant-antioxidant balance in asthmatics. Fifty six patients were divided into two groups: 1) placebo group, patients on standard therapy and 2) vitamin E-supplemented group, patients on standard therapy plus 400 I.U. Venous blood was collected on day 1 as baseline, then again after 8 weeks of respective treatments. The present study showed that standard therapy as well vitamin E-supplemented group had lower levels of superoxide anion generation as compared to the baseline. Plasma glutathione peroxidase (GSH-Px) was increased in standard therapy group whereas no difference was found in plasma GSH-Px from baseline in vitamin E-supplemented group. Plasma lipid peroxides were increased and total antioxidant capacity was decreased in standard therapy group whereas vitamin E-supplemented group had significantly lower levels of lipid peroxides and higher total antioxidant capacity. Total blood glutathione was also decreased in standard therapy group whereas no significant difference was found in vitamin E-supplemented group. Plasma nitrates and nitrites (NOx) were decreased in standard therapy group whereas they were increased in vitamin E-supplemented group. Plasma protein sulfhydryls and red cell superoxide dismutase (SOD) levels were increased in standard therapy group, while there was no change from baseline in red cell SOD activity in vitamin E-supplemented group, the levels of the former remained increased in this group also. No significant difference was found in plasma protein carbonyls and red cell catalase in either standard therapy group or vitamin E-supplemented group. Plasma vitamin E levels increased more than two fold after vitamin E supplementation but no change was observed in standard therapy group. There was significant improvement in FEV1% predicted in both the groups after 8 weeks of respective treatments but vitamin E-supplemented group had greater degree of improvement in terms of % increase from baseline. Our study provides biochemical and clinical evidence for the first time that vitamin E augments endogenous antioxidant screen and improves lung function. So, it may be used as an adjunct therapy in the treatment of asthmatics. RIC regimens (including highly immunosuppressive nonmyeloablative therapy in replacement of high dose chemotherapy or radiotherapy) for allo-SCT, are being explored with good results concerning feasibility and engraftment. However, little is known about the immune recovery pattern in these patients, especially the different CD4 and CD8 lymphoid T cell subsets. Here, we assessed at different time points after allo-SCT, the kinetic of recovery of naRve (CD45RA+/CD27+), central memory (CD45RA-/CD27+), and terminally differentiated (CD45RA+/ CD27-) CD4+ and CD8+ T lymphocytes in 64 patients from a single center, receiving HLA-identical RIC allo-SCT. Patients and graft characteristics are: age 48 y (27-63), diagnoses: 22 myeloid malignancies (34%), 22 lymphoid malignancies (34%) and 20 metastatic solid tumors (31%). 51 pts (80%) were considered as high risk. 91% of patients received a peripheral blood stem cell graft, with 42 (66%) receiving cyclosporine (CSA) alone for GVHD prophylaxis and 22 (34%) receiving CSA and MMF. In this series, in contrast to CD4+ T cell subsets, CD8+ T cell subsets had a progressive and sustained recovery in the first 3 months after allo-SCT, with acquisition of functional markers such as 2B4 and perforin. Among the different subsets analyzed, the recovery of naRve CD4+ T cells, and central memory CD4+ and CD8+ T cells, measured at day 28 after allo-SCT and before onset of grade 2-4 acute GVHD, showed a significant correlation with the risk of grade 2-4 acute GVHD (P = 0.001; P = 0.002 and P = 0.05 respectively). Patients developing grade 2-4 acute GVHD recovered a median of 47 naRve CD4+ T cells/AL prior to onset of GVHD as compared to 11 cells/AL in patients with grade 0-1 acute GVHD. NaRve CD4+ T cell levels significantly decreased after appropriate acute GVHD treatment. In a Cox multivariate analysis taking into account all relevant risk factors for acute GVHD, early recovery of naRve CD4+/CD45RA+/ CD27+ T cells in the first month after allo-SCT was the strongest parameter significantly predictive of grade 2-4 acute GVHD development (P = 0.006; Rr = 4.0; 95%CI, 1.5-11.0). Interestingly, there was a significant correlation between the total number of CD4+ T cells infused with the allogeneic graft and the early recovery of naRve CD4+ T cells (P = 0.001), suggesting that graft manipulation might represent an attractive tool towards harnessing alloreactivity after RIC-allo-SCT. CD8 T lymphocytes (CTL) play a major role in mediating allograft rejection in MHC-identical solid and bone marrow transplant settings. In such instances, alloreactivity is directed towards either donor-(solid organ) or recipient-(bone marrow) derived antigens that often represent only minor variations of self. These variant minor H antigens elicit robust CTL responses that in the most severe cases lead to graft versus host disease (GVHD) or graft rejection, and may result in death. The molecular mechanisms by which minor H antigens are processed and presented to donor or recipient CTL remain poorly understood. In this study, we have exploited mice deficient in various aspects of antigen presentation to demonstrate the roles of both donor-and recipient-derived dendritic cells and proteasomes in the aquisition, processing, and crosspresentation of minor H antigens. Our findings reveal an important role for recipient dendritic cells and donor proteasomes in the generation of an effective minor H antigen response. These findings have important implications not only for the understanding of GVHD, but for viral and tumor vaccine design as well. L. Stephan, 1 J. P. Tremblay. Bone Marrow or Stem Cell Transplantation Duchenne muscular dystrophy (DMD) a fatal neuromuscular recessive disease is characterized by widespread muscle damage throughout the body. Our research group is pursuing a research program to develop a treatment based on healthy donor myoblast transplantation (MT). However, the major draw back of MT is that long-term use of immunosuppressive treatments is associated with adverse effects: nephrotoxicity, increased cancer risk etc. .During last years, transplantation tolerance has been obtained by development of stable donor-specific chimerism resulted by bone marrow transplantation (BMT). Induction of stable multilineage chimerism (SMLC) across fully MHC-mismatched barriers have been established by a busulfan myelosuppressive treatment in mice primed with an allogenic spleen cells transfusion followed by a single cyclophosphamide (Cyp) dose, an immunosuppressive drug. We tried to obtain similar chimeris level in mdx mice, a dystrophic mouse model, with Cyp/Treo based protocol in order to prevent rejection of allogeneic healthy Balb/c myoblasts and of the muscle fibers that they formed in grafted tibialis anterior (TA).All mice (9) treated, have variable mixed chimerism levels (0.5-55%) for leukocyte cells population (CD90) 230 days after the BMT. Most (5/9) treated mice have CD90 chimerism between 10 and 20%. All treated mice have variable chimerism level in (CD4+ or CD8+) T-cell population in same proportion as CD90 population. Dystrophin positive fibers were present in chimeric TA mice 100 and 200 days after MT. Hybrid fibers number was equivalent to the one observed in TA sections of mdx mice treated with FK506 immunosuppression. No infiltration with CD4, CD8 T-cells was observed around dystrophin positive fibers at 100 or 200 days after MT. To test tolerance ddresistanceTT capacity, we have done a challenge by initially grafting donor myoblasts in mice left (6) TA using Cyp/Treo protocol developed above. One hundred days later, a second MT from the same donor strain was performed in the right TA without any additional therapy. Mice were sacrificed 100 days after the second MT. In both TA grafted, we observed dystrophin positive fibers and no CD4 or CD8 T-cells infiltration. Thus, we conclude that first grafts can survive after challenge with donor antigen/MT without additional treatment.Taken together, we show that Treo low toxicity associated with a protocol which does not require any irradiation and using only clinical use approved drugs, would permit to obtain safe sustained immunological tolerance of the DMD patients for donor MT. Accordingly, it could be applied as a conditioning regimen for several other organ or tissue transplantations. Neonatal CD4+ CD25+ T Cells-Age Restricted Development of Immune Tolerance. specialised T cells. The CD4+ CD25+ T cell is the best defined subset with regulatory function. Being essential in maintaining immune balance in mice and having shown regulatory capacity in man, the prerequisites for their development are still controversial. However, understanding the developmentally needs for immune tolerance might facilitate its manipulation.Having shown that the neonatal phase is pivotal to induce dominant tolerance to peripheral antigens, we built up a syngeneic bone marrow transplantation model to determine whether development of regulatory T cells is restricted to a certain developmentally window.For that reason, rag-/-mice were transplanted with T cell depleted rag F bone marrow. After reconstitution, non-irradiated recipients showed immune dysregulation and died of wasting disease. Disease was driven by host derived T cells. Bone marrow derived T cells abolished disease, whereas TCR transgenic or neonatal recipients were not protected. Depletion of CD4+/ CD25+ cells alone resembled whole T cell depletion, whereas addition of bone marrow derived CD4 cells prevented, or even cured, disease up to 30days after BMT. Strikingly, T cells developed in bone marrow chimeras were ineffective. Taken together, these results demonstrate, that regulatory T cells might be helpful tools treating immune dysregulation. However, their development is restricted to a certain developmentally window in early live while its largely diminished in adults or after BMT.Understanding development and role of regulatory T cells in immune reconstitution will allow us to improve GvT reactions while avoiding GvHD. We have previously demonstrated that anti-host CTLs characteristic of acute GVHD in the B6-into-F1 model are impaired with selective blockade of either IL-2 or IFNg. Surprisingly, preliminary experiments indicated that at 14 days after donor transfer, mice receiving combined IL-2 and IFN-g blockade exhibited more severe lymphopenia than untreated acute GVHD suggesting a paradoxical worsening of disease when compared to selective blockade of either IL-2 or IFN-g alone. To address the mechanism involved, acute GVHD was induced in B6D2F1 mice by the injection of 50 million B6 wild type (WT) parental spleen cells or 50 million B6 IFN-g -/-donors plus neutralizing anti-IFN-g mAb (XMG-6, 1 mg i.v. At 7, 10 and 14 days after parental cell transfer, mice were assessed for splenic lymphocyte subpopulations by flow cytometry and for ex vivo anti-host CTL activity. Our results indicate that peak donor CD8+ T cell expansion (day 10) is 2-fold greater for GVHD mice receiving combined IFN-g and IL-2 blockade compared to untreated or control mAb treated WT GVHD mice. Importantly, combined IFN-g and IL-2 blockade accelerated GVHD phenotype with peak anti-host CTL activity seen at day 7 (vs. day 10 for WT GVHD) and complete host B cell elimination and CTL downregulation seen at day 10 (vs. day 14 for WT GVHD). Of note, at day 7, GVHD mice receiving combined IFN-g and IL-2 blockade did not differ from IFN-g only blockade GVHD mice with respect to donor T cell engraftment or host B cell elimination. These results support the hypothesis that the early absence of IFN-g accelerates CTL development regardless of the presence of IL-2. In contrast, the absence of IL-2 after day 7 permits greater CD8+ T cell expansion, consistent with the loss of an IL-2 dependent regulatory mechanism. Extracorporeal photopheresis (ECP) is an immunomodulatory cell therapy being investigated as a treatment for various immune-mediated inflammatory disorders. Clinically, ECP involves the intravenous reinfusion of autologous, apoptotic peripheral blood leukocytes. In animal models, delivery of apoptotic cells has been shown to regulate immune responses through the modulation of cytokines, generation of regulatory T cells, and downregulation of antigen-presenting cell (APC) function. We and others have shown that activation of naRve T cells in the presence of APCs that have engulfed ECP-treated apoptotic cells leads to the generation of a T cell population with suppressive activity. In the present study, we demonstrate that the direct interaction of ECP-treated peripheral blood mononuclear cells (PBMCs) with naRve human CD4 + T cells in vitro promotes a T cell phenotype with regulatory activity. The generation of human regulatory T cells by ECP is dependent on APCs and can be reversed by the addition of IL-2. CD25 expression is upregulated on these regulatory T cells and they proliferate in response to concanavalin-A. However, these regulatory T cells do not express increased levels of Foxp3 or IL-10, nor do they secrete significant levels of the pro-inflammatory cytokines IL-2, IFNg, IL-4, and TNFa. Transfer of ECP-derived regulatory T cells to a secondary mixed lymphocyte reaction results in a greater than 50% inhibition of syngeneic responder T cell proliferation and IFNg production at a Treg:responder ratio of 1:20. In addition, transwell assays demonstrate that inhibition of responder T cells by ECP-derived regulatory T cells is contactdependent. Additional studies are underway to further characterize the phenotype of these regulatory cells and to determine their suppressive capacity in vivo. Our laboratory has shown that transplantation of hematopoietic stem cells (HSCs) into fetal sheep results in long-term multi-lineage hematopoietic chimerism. This observation demonstrates that tolerance can be induced during prenatal period by early injection of HSCs (optimal age for transplantation = 55-65 days of gestation, term = 145 days). However, the relationship between hematopoietic chimerism and tolerance has remained obscure. In order to understand the ontogeny of immune system in fetal sheep, we analyzed various cell populations in the thymus, spleen, bone marrow (BM), small intestine (site of PeyerTs patch formation), liver, and peripheral blood (PB) of fetal sheep, between days 39 to 82 gestation, by flowcytometery. These observations demonstrated that at day 39 the thymus contains up to 9% CD4+25+ cells. This population is minimally detectable at days 45 and 52 (0-1%); however increases to 11% at day 58 and then diminishes to 1-2% by day 65 and 2.5% at day 82. At day 58 of gestation, there is a peak CD4+25+ cells population in PB, BM, and liver as well. CD4+25+ cells are seen in BM, small intestine, and spleen at day 39 of gestation but not liver and PB. However, in BM and spleen, CD4+25+ expression diminishes to 2% at day 82 of gestation.The CD4+25+ cell population in peripheral lymphoid organs, at early gestational ages, express CD45R, likely a memory phenotype of T cells (as described before by Cooper et al. Journal of immunology, 1994, 152: 3098) . Immunohistochemistry studies at day 58 show CD45R cells abruptly increase in the medulla of the thymus, but decrease at day 65 and gradually increase at day 82.These data suggest that there is a population of CD4+25+45R+ cells (phenotypically consistence with T regulatory cells) in the primary and secondary lymphoid organs early in gestation. These data support the establishment of peripheral tolerance early in gestation that may have a role in tolerance induction following in utero HSCs transplantation. BACKGROUND Surprisingly, anti-tumor responses can occur in patients who reject donor grafts following nonmyeloablative hematopoietic cell transplantation (Dey et al., Biol Blood Marrow Transplant 7:604) . In murine mixed chimeras prepared with nonmyeloablative conditioning, we previously showed that recipient leukocyte infusions (RLI) induced anti-tumor responses against host-type tumors (Rubio et al. Blood 102:2300) .To further investigate the clinical relevance of this RLI model, we:1. Compared RLI with allogeneic lymphocyte infusion in untreated mice.METHODS Mixed chimerism was achieved in BALB/c (H-2 d ) mice conditioned with depleting anti-CD4 and CD8 mAbs on Day-5, cyclophosphamide 200 mg/kg on Day -1 and 7 Gy thymic irradiation on Day 0 prior to transplantation of 25 Â 10 6 B10.BR (H-2 k ) bone marrow cells. Some groups received RLI (3Â10 7 BALB/c spleen cells) seven weeks post-BMT. Some RLI donor mice received BALB/c A20 B cell lymphoma cells (1 Â 10 5 ) two weeks before RLI. Some groups received RLI depleted of B cells by MACS column for purging tumor cells. A20 cells (5 Â 10 5 ) were given i.v. one week after RLI in chimeras or after allogeneic lymphocyte infusion (3 Â 10 7 B10.BR spleen cells) to untreated BALB/c mice. RESULTS In the clinical setting, RLI would be obtained from tumorbearing hosts. We therefore examined whether RLI is still effective when the lymphocytes are obtained from tumor-bearing mice. Thus, with a purging procedure, the same anti-tumor effect was achieved with RLI from tumor-bearing hosts as from non-tumorbearing hosts.Allogeneic lymphocyte injection is a potentially feasible antitumor therapy. We therefore compared anti-tumor effects of allogeneic lymphocyte infusion into naive mice with that of RLI given to mixed chimeras. RLI recipients had longer survival than naive mice receiving allogeneic lymphocytes. This result suggests not only that the anti-tumor effect of RLI therapy is stronger than allogeneic lymphocyte infusion therapy but also suggests that rejection of allogeneic cells is insufficient and mixed chimerism is required prior to the induced rejection to achieve maximum antitumor effects.CONCLUSION Together, these data reinforce the potential of RLI therapy to be a new HSCT strategy which does not have the risk of graft-versus-host disease. ThymoglobulinR, a rabbit anti-human thymocyte globulin, is FDA approved for use in acute transplant rejection. Positive results in this setting suggest that ThymoglobulinR would likely be effective against graft-versus-host disease. To test this hyothesis preclinically, we generated an anti-mouse thymocyte antiserum (mATG) by immunizing rabbits with mouse thymocytes and purifying IgG from the resulting immuneserum. These methods were performed identically to those used to generate ThymoglobulinR, the human counterpart ATG. In addition, a model of acute graft-versus-host disease (GVHD) was developed. GVHD was induced by transfer of spleen cells from C57BL/6 mice into non-irradiated BALB/c RAG-2 deficient mice that lack mature T and B cells. Following transfer of the allogeneic cells, the C57BL/6 hosts displayed progressive weight loss which was accompanied by increased production of a variety of proinflammatory cytokines detected in the serum, hepatic necrosis and inflammation in several organs. In the absence of therapeutic intervention, control mice display significant disease within 3 weeks. We then applied mATG at various time points following cell transfer in the GVHD model to evaluate its effectiveness in disease prevention/reversal. Administration of mATG at days 0-3 post allogeneic cell transfer depletes most T cells from the circulation (FACS analysis) and completely prevents all evidence of acute GVHD. GVH mice treated with mATG showed no evidence of weight loss, elevated cytokine levels or target organ pathology. This protective effect of mATG on acute GVHD persisted for at least 12 weeks. We also tested the effectiveness of mouse ATG treatment given after acute GVHD was well established. In contrast to mATG administration at the initiation of disease, treatment 10-13 days following allogeneic cell transfer showed no evidence of GVHD amelioration. These results suggest that exploration of ThymoglobulinR dosing in relation to bone marrow transplant in human patients as prophylaxis for GVHD is warranted.This work is supported by Genzyme Corporation. The role of IFN-g in the induction of graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) has been elusive. IFN-g is protective in lethally irradiated mice receiving allogeneic T cells plus marrow cells, but deleterious in non-conditioned or sublethally irradiated recipients of allogeneic T cells alone (i.e., without marrow cells). We have recently observed that T cells from IFN-g KO donors induce more severe damage in parenchymal GVHD target tissues, but have reduced ability to eliminate recipient hematopoietic cells in lethally irradiated murine allo-HCT models. These results suggest that the deleterious effect of IFN-g in allo-HCT recipients of donor T cells alone might be due to facilitation of anti-host lymphohematopoietic GVH reactions, which result in destruction of host type hematopoietic cells and hematopoietic failure of the recipients. To test this hypothesis, we compared the development of GVHD in sublethally irradiated (6 Gy) B6D2F1 mice that received splenocytes alone or along with bone marrow cells (BMC) from IFN-g KO or wild-type (WT) B6 donors. B6D2F1 mice that received sublethal irradiation alone were used as non-GVHD controls and all survived long-term. Consistent with published studies, the recipients of WT B6 splenocytes alone (without BMC, so that the inoculum contained minimal numbers of hematopoietic stem cells) developed more severe GVHD compared to mice receiving a similar number of splenocytes from KO B6 donors (P b 0.05). In contrast, when mice received donor spleen cells along with BMC, the recipients of KO allo-HCT developed severe acute GVHD and succumbed to death by 5 weeks, while most of the recipients of WT allo-HCT survived long-term (P b 0.01). In order to quantitatively evaluate the ability of WT vs. IFN-g KO donor T cells to induce lethal GVHD, we compared the survival of lethally irradiated B6D2F1 mice that received a fixed number of BMC along with titrated numbers of splenocytes from WT or IFN-g KO B6 mice. The minimal number of splenocytes needed to induce lethal GVHD was approximately 10 fold less for KO than WT donor cells. Taken together, our results indicate that WT allo-HCT mediates stronger lymphohematopoietic GVH reactions than GKO allo-HCT, but the latter cause more severe damage in parenchymal tissues. Since lymphohematopoietic GVH reactions may selectively eliminate host lymphohematopoietic cells, including lymphoma cells, a better understanding of the mechanisms by which IFN-g separates the two types of GVH reactions could lead to novel approaches to separating GVHD and graft-vs.-leukemic effects. Background: It has been shown that CTLA-4 blockade early after murine MHC-disparate allogeneic bone marrow transplantation (BMT) results in augmentation of donor or host alloreactivity (graft-vs-host disease (GVHD), resp. Later after BMT, CTLA-4 blockade in combination with DLI induced a graft-vsleukemia (GVL) effect with a slightly increased risk for GVHD. Materials & Methods: C3H Â AKR 7,5 Gy radiation BM chimeras were treated with blocking anti-CTLA-4 MoAb (UC10-4F10) or irrelevant Hamster IgG, early (d-1 to 12) or late (d21 to 34). Mice were monitored for weight changes, clinical signs of GVHD and survival. Moribund animals were sacrificed for histopathology. FACS was used to study T cell chimerism and host/donorreactive T cell-frequency, MLR to study ex vivo alloreactivity, ELISA to determine circulating antiDNA Ab. Results: CTLA-4blockade early after non-T cell depleted (TCD) allo BMT induced acute and (N90%) lethal GVHD (increased donor T cell chimerism and alloreactive TCR-Vb6 + cells), histopathologically confirmed. Late after non-TCD allo BMT, however, CTLA-4blockade induced weight loss, alopecia, splenomegaly, lymphadenopathy and cachexia with 60% mortality. Treated and nontreated animals did not show any difference in chimerism or alloreactive T cell-frequency. Histopathology showed lymphoproliferation in spleen, lymph nodes but also in stomach, intestine, salivary glands and liver. In the liver, periportal infiltrates showed numerous plasmacells. AntiDNA Abs and liver-enzyme abnormalities were detected in the treated group. Ex vivo, splenocytes of diseased animals showed strikingly strong spontaneous proliferation but MLR reactivity towards host-or donor-type antigens was equal to that of tolerant non-treated chimeras. Conclusions: In a miHC-disparate context, CTLA-4-blockade, early after non-TCD BMT induces vigorous acute GVHD, consistent with activation of alloreactive T cells from the BM inoculum. In contrast, CTLA-4 blockade from day 21 onwards does not give rise to the breaking of transplantation tolerance, however, induces a lymphoproliferative syndrome with autoimmune manifestations.Our data suggest that after non-TCD allo miHC-disparate BMT, donor-host tolerance relies on clonal deletion which is insensitive to aCTLA-4 Ab. In contrast, tolerance to self is maintained by peripheral tolerance mechanisms, in which CTLA-4 signaling appears to play a major role.We are currently investigating if CTLA-4 blockade can elicit antileukemic reactivity and how the balance between autoimmunity and antitumor immunity can be modulated. 1 1 Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan; 2 Pediatrics, Fujita Health University, Toyoake, Aichi, Japan. Distinct Effects of Early and WHIM syndrome is a rare primary immunodeficiency disease characterized by Warts, Hypogammaglobulinemia, Infections and Myelokathexis. Until now, there is no report on a patient with WHIM syndrome treated by allogeneic bone marrow transplantation (BMT). We performed nonmyeloablative BMT for an 18-yearold female patient who has a nonsense mutation (S338X) in the chemokine receptor CXCR4 which was identified as causative gene of WHIM syndrome, from her healthy HLA-matched sister who has no mutation of the gene. The patient had experienced recurrent middle ear, sinopulmonary, and urinary tract infections since 2 years of age. She was recognized as leukopenic (white blood cell counts of 1.4 Â 10 9 /L), neutropenic (0.7 Â 10 9 /L) and hypogammaglobulinemic (IgG428mg/dl, IgA4mg/dl, IgM21mg/ dl), when she was admitted to a local hospital at 7 years of age with the aim of operation for chronic otitis media. She was referred to our hospital for investigation of leukopenia. Examination of bone marrow aspirate revealed a marked granulocytic hyperplasia and bone marrow neutrophils contained extremely pyknotic nuclei and vacuolated cytoplasms. Her disease was diagnosed as WHIM syndrome. Treatment with daily subcutaneous injection of recombinant human G-CSF increased mildly her blood neutrophils and decreased frequency of her febrile episodes. Four months later, therapy was discontinued because of the development of splenomegaly and myelofibrosis. Because her deafness was gradually aggravated, she was considered for allogeneic BMT at 18 years of age. This treatment plan was approved by the Institutional Review Board at Nagoya University Graduate School of Medicine and written informed consent was obtained from the patient, her parents and her sister. Because of the incompatibility between red-cell groups, a total of 30 Â 10 7 /kg erythrocytes-depleted bone marrow mononuclear cells were infused to the patient from her sister on January 16, 2002. GVHD prophylaxis consisted of tacrolimus and short-term methotrexate. The absolute granulocyte count in the peripheral blood was more than 500/Al on day 27. Although there was no evidence of infection or no sign of acute GVHD, mild eczematous lesions were observed on the body on day 70. She had N99% donor cells in peripheral blood as determined by microsatellite typing on day 30. Thirty six months after BMT, she is doing well, with normal blood cell counts and no need for therapy. Because allogeneic BMT has the potential to cure primary immunodeficiency diseases affecting marrow-derived cells, we consider that nonmyeloablative BMT is the treatment of choice for patients with WHIM syndrome who have an HLA-matched sibling donor. Human leukocytes respond vigorously when they encounter murine cells. One outcome of this response is the development of xenogeneic GVHD when human PBMNC are used to reconstitute immunodeficient recipient mice. It was hypothesized that preventing xenogeneic GVHD would permit the long term reconstitution of immunodeficient mice with functioning human cells. Two proposed approaches to prevent xenogeneic GVHD were depleting the xenoreactive cells prior to injection of PBMNC into the recipient mice or by generating recipient mice that lack the antigens that stimulate the xenogeneic GVHD responses by the human leukocytes. To characterize the human anti-murine response, the ability of unfractionated human PBMNC to proliferate in response to murine C57BL/6 stimulator cells lacking expression of murine H-2 class I, II or both was tested. The absence of H-2 antigen expression on the murine stimulator cells did not interfere with the proliferative responses of human PBMNC to these stimulator cells. These findings suggested that a number of human leukocyte subpopulations were able to proliferate in response to antigens other than H-2 antigens on xenogeneic murine cells. To characterize this response further, the cell surface markers expressed by activated CD69+ human leukocytes after incubation with murine stimulator cells were analyzed. These results indicated that the activated population of cells included T, B, NK, and NKT cells. Depletion of these activated CD69+ cells resulted in the inhibition of proliferative responses to the original C57BL/6 stimulating cells upon restimulation of the cells remaining after depletion. However the depleted cells were still able to proliferate to xenogeneic stimulator cells from other mouse strains and to allogeneic PBMNC. Additional studies will characterize the antigens responsible for inducing each of the responding lymphoid populations and test if administration of depleted populations prevents the development of xenogeneic GVHD.dysfunctions. During normal pregnancy, a predominance of Th2 type cytokines prevails and is considered to protects the foetus, while an increase of Th1 type cytokines may have deleterious effects. In this study we wanted to evaluate the possible role of certain Th1 and Th2 cytokines in the development of IUGR. We analyzed the gene expression, gene polymorphisms and protein levels of these cytokines from IUGR and non-IUGR pregnancies in a Pakistani population, where IUGR is very common.Material and methods: 45 IUGR and 55 control mother/infant pairs were studied. mRNA expression levels for IL-10, IL-8, TNFa, TGF-h, IL-6, IL-4, IL-1h, IL-12 and INF-g from the maternal (decidua) and the foetal (trophoblast) side of the placenta were quantified with RT-PCR. Cytokine and cytokine receptor gene polymorphisms for -1087IL10, -308TNFA, -174IL6, TGFB1, IL1RA, TNFR1, IL4RA 150V and -159CD14 were determined from genomic DNA by the combination of PCR and restriction enzyme cleavage. Serum levels of IL-1h, IL-6, IL-8, IL-10, IL-12, TNF-a and TGF-h were quantified in maternal and umbilical cord blood by ELISA and Luminex.Results: There was a significant decrease of IL-10 (P b 0.0001) and IL-12 (P b 0.008), but an increase of TGF-h (P b 0.009) in the decidua and similarly a decrease of IL-10 (P b 0.03), but an increase of TGF-h (P b 0.009) in the trophoblasts of the IUGR placentas compared to the non-IUGR placentas.We found significantly lower levels of IL-1h in serum from the mothers of the IUGR infants (P b 0.008) and of TGF-h in serum of the infants with IUGR (P b 0.05) compared to the non-IUGR infants.Conclusion: We note that the IL-10 mRNA expression in the decidua was down-regulated, but the TGF-h mRNA up-regulated in IUGR placentas of mothers from a population with multiple risk factors for IUGR. Background: Infertility affects 10% to 15% of couples desiring children. Immunologic factors have been proposed to be Involved in as many as 20%of otherwise unexplained cases of infertility. Due to Importance of antisperm antibodies diagnosis in Immunoinfertility, our main aim in this study is setup of IgG MAR and determination of the prevalence of ASA in zanjan province-Iran.Methods Introduction: Endometrial growth seems to be dependent on uterine artery blood flow and the importance of endometrial development on pregnancy outcome has been previously reported. Sildenafil citrate (VIAGRA), a type 5-specific phosphodiesterase inhibitor, augments the vasodilatory effects of NO by preventing the degradation of cGMP. Vaginal sildenafil improves uterine artery blood flow and sonographic endometrial thickness in patients with prior failed assisted reproductive cycles due to poor endometrial response. While improving uterine blood flow in the proliferative phase, NO may have detrimental effects at the level of the endometrium during the implantation window. The NOmediated release of cytokines such as tumour necrosis factor-from activated natural killer cells has been implicated as a cause of implantation failure. Therefore, the purpose of the study was to establish the effect of sildenafil on NK cell activity in women with a history of RSA, including women with multiple in-vitro fertilization failures. Materials and Methods: Fifteen nonpregnant women with the history of RSA and ten normal healthy women with the previous successful pregnancy outcome were studied. Measurement of uterine artery blood flow (pulsatility index, PI) was recorded using Doppler ultrasound by intravaginal probe in the study women. Natural killer cell activity was measured using flow cytometry. The following peripheral blood NK cellsT surface antigens: CD16, CD56 were also studied using flow cytometry. NK cell activity before and after sildenafil therapy in RSA women were studied. In addition, influence of 10mg or 400 ng sildenafil on NK cell activity after in vitro culture were performed. Results: We determined that there is a positive correlation between increased natural killer cell activity and PI in women with the history RSA compared to normal healthy women (r N 0.5, P b 0.05). Our preliminary data suggest that sildenafil has no significant influence on NK cell activity. Conclusions: Our data suggest that increased natural killer cell activity can be associated with diminished uterine artery blood flow. Sildenafil might be interesting therapeutic option for women with reproductive failure. However, further studies are needed to determine the role of this therapy in human reproduction. Objective: High level serum IgE is the hallmark of immune response in allergic patients. Epidemiological studies indicate that the prenatal environment plays an important and decisive role in the development of allergy later in life. To test whether the impact of maternal atopy could be transmitted through the maternal-fetal interface, this experiment was conducted. Methods: Animal model was developed by administration of allergens before mating for the study of the impact of maternal allergy on the development of an allergic immune response in early life. Healthy pregnant women who have approved to have their placenta collected for medical investigation are invited to give birth at the university hospital. Twenty-five pregnant women who were demonstrated to be atopics, based on clinical symptoms of atopic disease together with a positive Phadiatop and/or skin prick test gave their birth at the same hospital. Placentas were collected both from mouse and human groups and kept at À70C until the preparation of slides was carried out. All slides were doublestained for the analysis with immunohistochemistry. To detect macrophages both in murine and human placentas, monoclonal antibodies, F4/80 and anti-CD14 Abs, were employed in immunohistochemical procedures. Results: CD14+ placental macrophages show an intensive positive reaction to anti-IgE monoclonal antibody. Surprisingly, there was no remarkable difference between healthy mouse and the atopic model mouse as well as the human counterparts on the distribution pattern of IgE in placentas. Conclusion: IgE is distributed on macrophages both in murine and human term placentas. The atopic mothers could not pathologically impose their babies by their high level serum IgE during the pregnancy. Between 1% and 3% of women in the United States suffer recurrent miscarriages; 50% to 70% of conceptions fail. We have recently identified a novel role for complement activation in antiphospholipid antibody-induced pregnancy loss (J Clin Invest 112:1644 , 2003 . We now test the hypothesis that complement activation is a necessary intermediate in the pathogenesis of recurrent spontaneous miscarriage. DBA/2mated female CBA/J mice are a well-studied model of immunologically-mediated peri-implantation pregnancy loss that shares many features with human recurrent miscarriage. Embryos from mating CBA/J females with DBA/2 males show an increased rate of resorption (30-40%), compared to rates of b10% observed in the control groups (CBA/J Â BALB/c) (P b 0.01). In contrast, there was no evidence of complement in decidua from CBA/J Â BALB/c. To provide direct evidence that complement is, in fact, a critical mediator in this model of spontaneous pregnancy loss, we attempted to rescue fetuses from DBA/2-mated CBA/J mice with complement inhibitors. To block C3 activation by alternative and classical pathways, we treated mice with a recombinant C3 convertase inhibitor Crry-Ig (3 mg ip day 2, 4 and 6). Inadequate expression of complement regulatory proteins is a possible cause of increased complement deposition and embryonic death, but Western blotting of embryos and deciduas did not show such deficiency.To examine the importance of complement activation at the level of C5, we treated DBA/2-mated CBA/J pregnant mice with anti-C5 mAb. To distinguish the role of C5a and C5a receptor (C5aR) from that of MAC seeded by C5b, we treated pregnant mice with a highly specific peptide antagonist of C5a receptor (C5aR-AP) AcPhe[Lornithine-Pro-D-cyclohexylalanine-Trp-Arg]. C5aR-AP (100 Ag ip on day 2) significantly improved pregnancy outcomes (8.0 F 5.9% fetal resorptions, P b 0.01) suggesting that C5a-C5aR interactions play a critical role in fetal loss. Given the importance of alternative pathway in ischemic and antibody-trigged injury, we considered the role of factor B in our model. These studies identify key innate immune effectors that mediate poor pregnancy outcomes and provide novel and important targets for prevention of recurrent pregnancy loss in patients. A fetus, although semi-allogeneic, is usually accepted by the maternal immune system. However, complications including alloresponsive mechanisms are thought to be potentially detrimental for a successful pregnancy. Therefore, we used the mixed lymphocyte culture (MLC) reaction to compare the allogeneic T-cell response of non-pregnant women (n = 96) with the response of healthy pregnant (n = 68) and pregnant women affected by different gestation-associated diseases such as prolonged preterm rupture of fetal membranes (PPROM) (n = 20), uncontrollable preterm labor (PL) (n = 15), preeclampsia (n = 22) and intrauterine growth retardation (IUGR) (n = 12). Peripheral blood mononuclear cells (PBMCs) of all three groups were stimulated with PBMCs from unrelated volunteers. Exposing PBMCs from pregnant women to PBMCs of their own fetus led to a further significant decrease of SIs (median value 2.2, range 0.6-51.5). Among the two groups of pregnant individuals, SIs of women with prolonged preterm rupture of fetal membranes (PPROM) were significantly higher (median value 6.3, range 1.3-56.8) in comparison to women with uncontrollable PL (median value 1.4, range 0.9-8.5) , women with preeclampsia (median value 1.8, range 1.1-14.9), women with IUGR (median value 1.9, range 0.7-38.1) and women with normal term delivery (median value 2.2, range 0.6-51.5) when the maternal PBMCs were stimulated with PBMCs of their own fetus. This phenomenon could not be observed after stimulation with PBMCs from unrelated volunteers. In addition, an increased humoral immune response was assessed for women with PPROM (40.5 % women anti-HLA-antibody positive) in comparison to women with uncontrollable PL (15.2 % women anti-HLA-antibody positive). Our results revealed a strongly reduced allogeneic T cell response of PBMCs from pregnant women that was further downregulated when PBMCs from their own fetus were used as stimulators. The same finding could be observed in pregnant women who suffer from different gestation-associated diseases. An exception were women with PPROM who exhibited a comparatively high alloresponse implying an increased anti-fetal reaction under these conditions. Objectives: The Signal Transducer and Activator of Transcription 3 (STAT3) is involved in the invasiveness of carcinoma cells. In an earlier study, we found that invasiveness correlates with STAT3-DNA binding activity in trophoblast and choriocarcinoma cells. Aim of this investigation was to verify the role of STAT3 in these observations by RNA interference induced STAT3 knock down. Methods: By using short interference RNA (siRNA), STAT3 was knocked down in the Jeg-3 choriocarcinoma cell line. Oligonucleotides were designed to interfere exclusively with STAT3 mRNA. For control scrambled oligonucleotides were designed to not interfere with any known human protein. The successful knock down was analyzed by polyacrylamid gel electrophoresis and western blot. Invasion of cells was measured by counting cells on the filter beyond the matrigel as well as in the medium beyond the filter. Results: By applying a concentration of 66nM of siRNA oligonucleotides the invasion of Jeg3 choriocarcinoma cells was reduced by 57%. Invasion of non-transfected cells was increased by stimulation with Leukemia Inhibitory Factor (LIF; 64%) and Interleukin-6 (IL-6; 12%). In STAT3-knock down cells these effects were completely blocked for IL-6 and significantly reduced for LIF (16% invasion). Conclusion: STAT3 plays a key role in the invasion of choriocarcinoma cells. The slight remaining invasive capacity of Stat3 knocked down cells may be due to a not complete elimination of Stat3 or to further invasion triggering pathways. Introduction: One of the most important problems in detection of antisperm antibody (ASA) is to design an internatinal correct method with at least false answers. It seems that Enzyme-Linked Immunosorbent Assay (ELISA) will be more sensitive, specific and with more diagnostic value for detection of ASA, if it is used sperm surface antigens without contamination with sperm inside antigens and nonspermic antigens as coated antigens in ELISA technique. In this way, some techniques such as using different specific detergents are recommended. This study has focused on different methods of sperm surface antigens extraction to determine a better method of antigens extraction for ELISA technique. Materials and Methods: We extracted sperm surface antigens by four different method: Sonication method, using NaCl salt, SDS detergent and using LIS detergent on sperm prior treated by biotin and then we evaluated all four extraction method by blotting and ELISA technique to assess the better exposing surface antigens. Results and Conclusion: Our results have demonstrated that extraction method by LIS detergent expose sperm surface antigens better than other three methods and we concluded that this method is superior for using in ELISA technique. F2 Other F2.09. The Changes of T Lymphocyte CD Markers in Patients Exposed to Mustard Gas. INTRODUCTION: The immunodepressive effects of mustard gas was detected in previous studies.The present study was performed on 31 Iranian chemical casualities, after 10 years of exposure to mustard gas, to determine the changes of absolute lymphocyte count and T cell CD markers compared to normal control group.METHODS: CBC and absolute lymphocyte count was determind by H 1 automatic system.T cell CD markers (CD2, CD3,CD5,CD7) was analyzed by Flow cytometric method.using conjugated monoclonal antibodies.RESULTS: Absolute lymphocyte count were increased but lymphocyte CD2.CD3 and CD5 markers were decreased significantly (P b 0.05) compared to normal control group (The decrease in CD4 and CD8 markers were reported by other investigators previously).CONCLUSION: The increase suseptibility to respiratory and other infections in these patients, could be due to decrease in T lymphocyte subsets.Increase in absolute lymphocyte count could be due to lymphoproliferative changes in B or NK lymphocyte lineage,and requires another investigation. CD147 displaying on VCSM13 phage was generated to enhance the functional affinity of CD147 for its partner (s) and to study the ligand-receptor signaling in U937 monocytic cell line. Cellular morphological change of U937 incubated with multivalent CD147 phages had been observed since 24 h of cultivation and cell propagation was ceased after 48 h. This phenomenon was presumably related to the anchoring of multivalent CD147 phage to U937 surface molecule (s) proven by either ELISA or immunocytochemistry. Interestingly, the apoptotic nucleus was found to coordinate with U937-bound phage. Cytotoxic activity of CD147 phage on U937 was further analyzed by EthD-1/calcein AM double stains. In contrast to wild-type phage, dual-colour fluorescent in U937 induced with recombinant phage, which associated with the apoptotic characteristic, was indicated. The level of cleaved caspase-3 in U937 incubated with multivalent CD147 phage was not as high as in U937 stimulated with cisplatin when determined by flow cytometry. Nevertheless, the signal was apparently stronger than uninduced U937 and VCSM13-incubated U937. Quantified by immunocytochemistry, only 12.8% of multivalent CD147-activated U937 with apoptotic nucleus harbored substantial amount of cleaved caspase-3, whereas 41% was resulted from cisplatin-induced U937. Accordingly, the caspase dependent pathway supposed to partially involve in CD147provoked cell death program. A novel function of CD147 in triggering apoptosis implied the existence of CD147 counterreceptor (s) on U937 membrane. Sensitivity of Soil Bacteria towards Cadmium Metal and Its Effect on Moung Bean Plant. Introduction Pollution is now a days a termendous threat to the universe, progress in different scientific fields helps at one side to the world but on technologist hand it causes great dangerous effects on human and plants. Heavy metal pollution (hanaeklaus., 1999) in aquatic system has become a serious threat today. Heavy metals are wide spread pollutants of great environmental concern as they are non degradeable and thus persistant (Trueby.P., 1990) Many bacteria are facultative anaerobes. Nitrogenfixing facultative bacteria are generally only capable of fixing nitrogen when they are growing in anaerobic environments. Examples of such bacteria include some of the Enterobacteriaceae, such as Enterobacter species (Camb. press).Chemical treatment of water resources are very expensive, in this endeavour,microbial biomass has emerged as an option for developing economic and ecofriendly waste water treatment process but presence of heavy metal can damage microbial life.The present study was carried out to check the toxicity of Cd (cadmium) metal on bean plant and their physiological process in relation with nitrifying bacteria in soil and roots and shoots of the plant.Methodology Plants were analysed after 15 days of germination, the contents of chlorophyll a, b carbohyderates, proteins and amino acids were determiend by standered method (Chapman.S.B.,1976) . Mineral ions were analysed by flame photometry and atomic absorption techniqe.The toxicity of different concentrations of Cd metal on nitrifying bacteria was determined by SPC (standard plate count) method. Results and discussion Cd was found to be highly toxic to the seedling as well as for the microbial life present in the rhizopsphere of bean plant at all concentrations used.Morphology of plant was effected while colour of plant turns yellow. The growth of the plant was inhibited and the length of root and shoot was found to be decreased, cmpared with controll plants.Reduction in the proceses of the photosynthesis was observed as yellow coloured leaves appeared instead of green coloured because of the decline in the chlorophyll a and b contents.Nutritive values of plants decreases with the increase in the concentration of Cd metal which may be due to the absence of nitrifying bacteria which are normally found in symbiotic association with these plants where they fixes atmosphereic nitrogen in the roots of leguminous plants ( Dakora and Donald, 2002) but in the presence of Cd metal nodule formation was inhibited results in the decrease in the protiens contents and absence of amino acids at high concentrations of Cd. Potassium and sodium (Hsiao.T.C.,1986)were investigated and low percentages showed that accumalation of Cd was higher in the roots as compare to the other mineral ions due to which plants were not able to stand in the errect position.Iron Magnesium Magnese which were responsible for metabolic activity, transpiration and translocation were also effected and their percentages were also found to be decreased at higher concentration of Cd. Introduction: The mannan-binding lectin (MBL) pathway of innate immunity is important in host defense against pathogens. Major surgical trauma is known to influence various immune functions and in the present study we investigate the effect of major surgery on two central components of the MBL pathway; MBL and the associated protease MASP-2, compared with the effect on IL-6 and CRP levels.Methods: Sixty patients were randomized to open or laparoscopic colectomy for benign or malignant disease. Serum levels of MBL, MASP-2, IL-6 and CRP were determined preoperatively, and 1, 2 and 6 hours following incision, and at postoperative day 1, 2, 8, and 30 .Results: All four parameters showed a slight decrease in serum levels within the first two hours after incision. For MBL and MASP-2 a minor, but significant (P = 0.01 and P = 0.04 respectively) peak was found on postoperative day 8. Compared to the preoperative level a significant, 10-fold increase of IL-6 was found 6 hours after incision (P = 0.0001), and with levels significantly lower on day 30 (P = 0.0005). For CRP a significant increase was seen on postoperative day 2 (P = 0.0001), whereas the levels on day 30 were not statistically different from preoperative levels (P = 0.08). The levels of IL-6 and CRP were significantly correlated (r = 0.71, P b 0.0001), whereas no other significant correlations were detected between the parameters. No significant differences between the responses to the two surgical techniques were revealed.Conclusion: In contrast to the marked effects on the levels of IL-6 and CRP major surgery only marginally influenced the MBL pathway. There was no difference in the response to the two different surgical techniques. Objective: To study the theory of hemaimmune reaction. Methods: Cancer cells or yeast cells (or NS) were added into human fresh anticoagulant whole blood (or blood cells and plasma) treated by citric acid, and incubated for 30 minutes at 378. Main Outcome Indexes: adhering rate. IL-8, CD35, DARC (Fy6), CXCR4 et al.Results: It was found that cancer cells (dead cells) and yeast cells can activate a war of hemaimmune reaction (HIR). In time of war against cancer cells and yeast cells, the level of indexes (adhering rate, IL-8, CXCR4) was significantly higher than that in time of peace (NS control group). In time of war against cancer cells and yeast cells, level (IL-8) of HIR in white blood cell group with plasma added was significantly higher than that in white blood cells group without plasma. Level (IL-8, CXCR4) of HIR in white cells group with red blood cells added was significantly higher than that (IL-8, CXCR4) in white blood cells group without red blood cells added.Conclusion: human hemaimmune reaction looks like a modern war in the body. The results suggest that the complement and red blood cell play a vital role in blood immune reaction, and there is a road map of hemaimmune reaction: Antigens (cancer cells or yeast cells) can activate complement in plasma, then the antigens which were opsonized by complement C3b are mainly adhering to red blood cells, and then they are adhering to white blood cells to activate hemaimmune reaction system (see Figure l) . Furthermore, it can provide useful information for studying innate and adaptive immune and for establishing experimental (or war type) system of hemaimmune reaction road map.F2.14. A Novel CD70+ APC Imprints a Unique Pattern of NK Receptors on Gut Mucosal CD8 T Cells. We have identified a novel antigen presenting cell population in the intestinal lamina propria that constitutively expresses the costimulatory molecule CD70. Here we show that stimulation via this APC induces a unique pattern of NK receptors on the mucosal CD8 T cells. Resident CD8 T cells in the gut mucosa in naRve mice exhibited an activated phenotype and expressed the Ig family NK receptors 2B4 and gp49B1 but did not express the lectin-like CD94-NKG2 heterodimeric receptor. In contrast, activated CD8 T cells in the peritoneal exudate lymphocytes and spleen following Vaccinia or Listeria infection expressed gp49B1 and CD94-NKG2 and but did not express 2B4. Similar differences in NKR expression were also found on antigen-specific CD8 T cells generated at different sites during the same infection. CD70 antibody treatment however, had no effect on NKR expression by activated CD8 T cells at peripheral sites, where CD70+ APC do not occur. However, when mice were intraperitoneally immunized with CD70 expressing allogenic P815 cells, 2B4 was induced on CD8 T cells accumulating in the peritoneal cavity. Thus, CD70 costimulation via CD70+ APC imprints unique NK receptors on mucosal T cells. We demonstrate the application of Bayesian networks for computational elucidation of causal intermolecular influences in signaling networks, using simultaneous multivariate measurements of phosphorylated proteins and phospholipids in populations of single human primary CD4+ T cells. Selective perturbations, both activating and inhibiting, were important to inferring the direction of influence between signaling components. We identified most classically reported signaling relationships and predicted novel influence connections, including inter-pathway crosstalk from the kinase Erk1 to the kinase Akt (confirmed experimentally). These results manifest the feasibility of data-driven construction of causal signaling network models from primary cell data at the single cell level and may have utility in understanding patient specific signaling alterations in disease states. Immunophenotyping of peripheral blood mononuclear cells (PBMCs) is of limited value for assessment of most clinical states. As a more informative alternative, immunophenotyping may be combined with functional assays as a correlate of clinical status. Most functional assays, however, are tedious or require prolonged culture periods. One simple, highly informative, and rapid approach is to examine cell signaling within individual cells following brief stimulation. Abnormalities within the signaling pathways of specific cell types could provide important insights concerning pathological conditions. This study focuses on the JAK/STAT pathway as it is central to host defense, cell growth, and apoptosis. Dysfunction of this pathway has been observed within cancer cells of various types. We have developed conditions allowing dual labeling of cell surface markers and intracellular phosphorylated STAT members within individual, normal PBMCs, which have undergone rapid cytokine stimulation in vitro.The PBMCs are first incubated with a primary antibody against cell surface CD4, CD8, CD14, or CD56. Cytokine [IFNg, IL-2, IL-4, or IL-13] treatment is then applied to induce STAT phosphorylation. The cells are immediately fixed with 2% PFA and then permeablized with a cocktail of saponin, methanol, ethanol, isopropanol, or acetone at varying concentrations. The cells are labeled with primary antibody against several pSTAT proteins. Analysis of the efficacy of extracellular and intracellular labeling is completed using a BD FACSArray flow cytometer.With all of the reagents tested, it appears that there is a trade-off between intracellular and extracellular labeling of cells. 90% methanol, which was previously used in our lab and in the labs of others, gives a good signal for phospho-STATs, but does not allow identification of many cell surface molecules. Other permeabilization methods, such as saponin, and lower alcohol concentrations, allow for better cell surface labeling, but simultaneously cause a decrease in the pSTAT signal. Of the reagents tested, 70% methanol allows for the best dual labeling of both intracellular and extracellular proteins. These methods will permit rapid analysis of complex cell populations, including PBMCs. Interrogation of signaling pathways in individual cell types will allow more profound evaluation to gain additional insight into abnormalities causing or arising from the presence of immune dysfunction. Dendritic cells (DC) are key regulators of innate and acquired immunity. DC maturation is a critical event in the DC life cycle, conferring to it the capacity to both simulate T cell proliferation and polarization. DCs may be matured by Toll receptor ligands (like LPS), as well as by T cell dependent mechanisms via CD40 ligandmediated (CD40L) activation. We have previously demonstrated that cigarette smoke extract (CSE) inhibits several maturation events induced by lipopolysaccharide (LPS), including the upregulation of co-stimulatory molecules, and production of the key Th-1 polarizing cytokine IL-12p70. In the current study, we hypothesized that CSE also impairs DC maturation mediated by T cell dependent pathways through CD40L stimulation. CSE was generated by bubbling the smoke from one cigarette (1R3F University of Kentucky) through 10 ml PBS. Immature DCs were generated from human monocytes cultured with IL-4 (5ng/ml) and GM-CSF (800U/ml). During the final 48 hours of culture, DCs were incubated with CSE at concentrations that do not diminish cellular viability (as measured by Annexin V and Propridium iodide staining). During the final 24 hours, DCs were matured with interferon-gamma (50ng/ml) and CD40L (500ng/ml). The expression of costimulatory molecules was determined by flow cytometry and IL-12p70 concentration was determined by ELISA. PGE 2 levels were also analyzed by ELISA. CSE-conditioned DCs matured with CD40L, expressed lower levels of CD-86 and CMRF-56 compared to control DCs. CSE-conditioned DCs produced more PGE 2 than controls, suggesting a mechanism by which CSE alters DC maturation. Our data illustrate that CSE alters DC maturation induced by both LPS and CD40L. The inhibition of IL-12 production by CSE illustrates an important mechanism by which smoking inhibits essential adaptive immune responses relevant to the pathogenesis of cancer and certain infections. Tanveer Khanum, Nadia Noor, Nasra Jalil, Sadaf Hedayat. 1 Microbiology, Jinnah University for Women, Karachi, Sindh, Pakistan; 2 Microbiology, Jinnah University for Women, Karachi, Sindh, Pakistan.Bacteriocin are low molecular weight single polypeptide or polypeptide complexes having an antibacterial activity, synthesized in ribosomes and secreted by bacterial cells or bthe group of heterogenous substances ranging from low molecular weight compounds to high molecular particles resembling bacteriophage protein components (Rasool & Khan, 1991) .Q In 1967 (Bradly) classified bacteriocins into two broad types: a) A group of low molecular weight, trypsin sensitive, thermostable bateriocin.b) A group of high molecular weight, trypsin resistant, thermostable beteriocin.Microorganisms in yogurt and the subsequent creation of organic acids and bio-active proteins inhibit the growth the growth of many pathogenic microorganisms.Methodology Different yogurt samples were used for isolation of bacterial strains After making the dilution of yougurt samples, the diluted samples were spreaded on nutrient agar and incubated at 37 8C. Isolated colonies were identified by using different conventional methods. Ten reference strains were used to check bacteriocinogenic activity. The isolates includes E. coli, Staph.aureus, Klebsiella, Salmonella, Enterococcus, Micrococcus, Bacillus, Staph. epidermidis, Pseudomonas. After this bacteriocin production was detected againt reference strains by using agar well and cross streak methods. The titres of bacteriocin produced were quantified by two fold serial dilutions of bacteriocin.Results and disscussion The average colony forming unit per ml calculated as 8.4Â10 6 Cfu/ml. The bacteria isolated were identified by using different conventional methods. Bacteriocin of Lactobacilli was active against Shigella, Bacillus and Micrococcus.The bacteriocin of bacillus was active against S. aureus, St. epidermidis, Enterococcus and E. coli.Bacteriocin of Pseudomonus was active against Shigella, S. aureus, St. epidermidis, Entercoccus, Micrococcus, Bacillus and S. typhi.The arbitrary unit of bacteriocin of Lactobacilli against Shigella is 1:4, against S. aureus is 1:2 against St. epidermidis is 1:8 against Entercoccus is 1:8, against Bacillus is 1:2.The arbitrary unit of bacteriocin of Pseudomonas against Shigella is 1:6, against S. aureus is 1:2, against St. epidermidis is 1:4.The arbitrary unit of bacteriocin of Bacillus was not found. It was found that yougurt contain high number of bacteria which include both Gram negative and positive species of bacteria. The bacteriocin of lactobacilli are broad spectrum as active against both Gram negative and positive bacteria and best inhibitory activity was observed against St. epidermidis.Pseudomonas produce powerful bacteriocin which inhibit the growth of majority of clinical isolates where bacteriocin of Bacillus and Lactobacilli were not as effective.We also observed those bacteria which produce bacteriocin line of diffusion after removal of growth and also bacteriocin activity was best observed when the plates were first refrigerated and then cross streaked which indicate that refrigeration temperature facilitated the diffusion of bacteriocin in medium.Brussels, Belgium; 3 Laboraory of Physiology, Vrije Universiteit Brussel, Brussels, Belgium.We previously showed that FasL retrovirally transduced bone marrow-derived DC increase allospecific cytotoxic activities and Th1 cytokines production in vivo thanks to a FasL dependent neutrophils recruitment. The aim of our study is to test if the expression of FasL by DC can promote a stronger cytotoxic activity and a better anti-tumoral immunity compared with DC expressing a control Ag. We first evaluated a therapeutic approach which consisted in subcutaneous injection of EG7-OVA tumor cells and FasL or control-DC in C57BL/6 mice. We realised histological analysis on tumor sites and we measured the anti-tumoral T cell response. We observed that co-injection of FasL-DC and EG7-OVA cells inhibit tumor growth. Histology of tumors sites harvested from mice co-injected with control-DC and EG7-OVA cells showed only a moderate inflammatory infiltrate composed essentially of mononuclear cells. In contrast, tumor sites coming from mice co-injected with FasL-DC and EG7-OVA cells reveal a massive destruction of the tumor associated with apoptosis and neutrophils recruitment. We demonstrated that in vivo EG7-OVA apoptosis could not be explained only by the tumoricid activity of FasL-DC since we observed that only 6 to 7% of tumor cells were lysed by FasL-DC in vitro. We demonstrated that neutrophils contribute to the tumor eradication in FasL-DC and EG7-OVA co-injected mice as anti-Gr1 monoclonal antibodies treatment restore tumor growth. Finally, mice coinoculated with FasL-DC and EG7-OVA tumor cells displayed a higher Th1 cytokines production and were protected against tumor challenge. Indeed, we observed that the antitumoral protective response was mediated by OVA-specific CD8 + T cells. We now evaluate the role of neutrophils recruited by FasL-DC in the induction of the OVA-specific T cell response. Tumor vaccines are targeting CD8+ T-cells capable of differentiating into activated effector cells which mediate tumordestruction, and memory cells crucial for long-term tumor-immune surveillance. The CD8ab heterodimer represents the predominant CD8 form in the peripheral blood circulation, although some CD8+ T-cells express the homodimeric CD8aa. Recent data suggest that CD8aa cells may represent a biologically important subset of memory T-cells. We tested longitudinally sampled peripheral blood mononuclear cells (PBMCs) from patients with melanoma, vaccinated with the differentiation antigen Melan/MART-1, for i) the presence of CD8aa expressing T-cells, ii) for T-cell differentiation and homing markers (CD45RA/CCR7) and iii) antigen-specific T-cells using AAGIGILTV (naturally processed peptide) and ELAGIGILTV (superagonist) peptides loaded into HLA-A2 tetramers. MART-1/Melan-A reactive T-cells were present in CD8ab and CD8aa expressing T-cells. Although CD8ab and CD8aa cells show a different composition based on CD45RA and CCR7 expression, the examination of tetramer Melan/MART-1 specific T-cells in the two CD8 subsets shows a terminally differentiated phenotype (CD45RA+/CCR7-) which is maintained over time. These data suggest that CD8aa cells represents a memory T-cell pool, contributing to a long-lived immune protection. We could consolidate this hypothesis using transfected recipient surrogate (TCR-/CD8-) cells which express either the MART-1/Melan-A specific TCR and CD8aa or CD8ab as a transgenes: TCR interaction with HLA-A2/peptide complexes shows different requirements in TCR+/CD8ab cells as compared to TCR+/CD8aa cells. In vitro culture of PBLs from vaccinated patients with melanoma, using different cytokines and peptide stimulation, showed that IL7 or IL2 lead to a differential expansion of the CD8aa and CD8ab Melan/MART-1 specific T-cell population, respectively. This represents the first ex vivo report of anti-tumor directed CD8aa T-cells in patients with melanoma undergoing peptide vaccination. The assesment of this memory T-cell population will be relevant for immunomonitoring of patients with tumors and aid to design improved tumor vaccines capable of stimulating long-lived cellular immune responses. Human Adipocyte and Its Participation in Innate Immunity.Laurence Hoareau, 1 Marie-Paule Gonthier, 1 Regis Roche, 1 Franck Festy, 1 Christian Lefebvre D Hellencourt, 1 Maya Cesari, 1 Jean-Pierre Riviere, 2 Marie-Amedee Dijoux, 2 Sandrine Bes-Houtmann. 1 1 University of Reunion, LBGM, Saint Denis, Reunion, France; 2 Laboratoire dTanapathologie, CHD Felix Guyon, Saint Denis, Reunion, France.Introduction: In addition to the well know role of adipocyte in energy homeostasis, some recent studies suggest that this cell is also involved in immunity. To further investigate the immune capacities of adipocyte, we studied the expression of Toll Like Receptors (TLR), which are important players in innate immunity. In addition, the functionality of TLR2 and TLR4 was tested, by using one of the TLR ligand in adipocytes culture.Methods: Human subcutaneous primary preadipocytes and mature adipocyte were used as cellular model. The presence of different TLRs was determined at RNA level by reverse transcriptase polymerase chain reaction (RT-PCR) and at protein level by Western Blotting and immunocytochemistry. Furthermore, cells were stimulated with lipopolysaccharide (LPS), and cytokines gene expression were quantified at different times by real time PCR, in order to assess adipocytes response to one of the TLR ligand.Results: We demonstrated that human subcutaneous adipose cells and tissue expressed TLR 2 and 4 mRNA. These results were confirmed by the presence of the corresponding proteins. The same results were observed in mature adipocytes and in the tissue. After stimulation with LPS, an increase in mRNA level for Cyclooxygenase-2 (Cox-2) and interleukine 6 (IL-6) was observed in culture.Conclusion: Our results showed for the first time the presence in adipocytes of TLR 2 and TLR4, two important receptors involved in innate immunity. These receptors are present not only on preadipocytes but also in mature adipocyte and on the adipose tissue. Furthermore, we demonstrate that these receptors on adipocytes are able to signal an immune response such as cytokine production. These results suggest that adipose cells could respond to bacterial infection via the interaction between bacterial component and TLR and participate in innate immunity. Finally, to better understand the mechanisms of adipocyte response to infection, analysis of TLR signalling pathways from subcutaneous adipocyte are in progress. HDL-Reverse Cholesterol Transport and Signal Transduction in T-Cells.T. 2 1 Research Center on Aging, Universite de Sherbrooke, Sherbrooke, QC, Canada; 2 Deapartment of Biochemistry, Universite de Sherbrooke, Sherbrooke, QC, Canada.HDL-mediated Reverse Cholesterol Transport (RCT) serves to regulate plasma cholesterol levels as well as cholesterol exchange with circulating cells. We have previously shown that membrane cholesterol in increased by 2-folds in T-cells from elderly donors. This was accompanied by defects in cellular activation leading to immune senescence. RCT is well-known in the case of atherosclerosis but its role in T-cells cholesterol metabolism as well as T-cells signalling is still unknown. RCT is a lipid rafts-dependent mechanism that involves a fast and a slow pool of membrane cholesterol. In this study we sought to determine the role of HDL in cholesterol metabolism of T-cells. We separated T-cells from young (b25 years) and elderly (N65 years) healthy SENIEUR subjects. We studied cholesterol uptake by the tritiated cholesterol and for extrusion by HDL-driven reverse cholesterol transport. The cholesterol uptake was decreased in T-cells of elderly. The fast cholesterol pool is extracted from lipid rafts microdomains, but separated lipid rafts from elderly donors still contain a high amount of cholesterol. The incubation of T-cells with HDL induced a signalisation that involved Jak-2 after 90 min that corroborates with the fast pool cholesterol (lipid rafts) while a long lasting activation of ERK is observed after 24h that corroborates with the slow pool cholesterol (non-rafts). In the case of elderly donors, the activation of these pathways is differentially altered. These data are the first to show that HDL-mediated RCT is impaired in aging explaining the changes in lipid rafts properties. Moreover, it has been suggested recently that lipid rafts play an important role in the cholesterol exchange mediated by HDL. Thus, lipid rafts may auto-regulate their cholesterol content and changes in lipid rafts properties with aging may explain defects in T-cell activation. The Effect of Treatment Recurrent Herpes Simplex with Larifan. of Dermatovenereology, Riga StradinTs University, Riga, Latvia; 2 Institute of Microbiology and Virology, Riga, Latvia.Introduction. Viral infection of herpes has captured a growing attention during the last decade that shall be explained with the ever-increase of herpes viruses throughout the world, frequent relapses and unsatisfactory results of the usually prescribed therapy of Herpes simplex viruses.Various researches deal with the immunology of herpes virus attempting to more accurately define the impact of immune regulation factors.The therapy of Herpes simplex infection more excessively suggest immune modeling remedies that directly or indirectly affect the response of immune competent cells.Aims of the research. To evaluate the efficiency and safety of Larifan rectal suppositories for the treatment of frequently relapsing infections of simple Herpes as well to detect the concentration of circulating interferon during therapy.Methods. The research compromises the analysis of a variety of Larifan-suppositories, a general and local antiviral medicine synthesized in Latvia. Larifan is a medicine of natural origin of doubly spiraled ribonuclein acid that forms up in Escherichia coli cells that are infected with bacteriofages. In vitro proves its capability to slow down the reproduction of encephalomyelitis in Venezuela horses and other viruses, reducing the output of viruses for 100 000 times. The animals involved in experiments proved to have both preventive and therapeutic affects in case of tick encephalitis, mice encephalomyocarditis, rabies, influenza and other viral infections.Out of the total number of 36 patients involved in the research, all of them had frequently relapsing simple herpes (6 times and more per year), 19% (n = 7) had genital herpes, 81% (n = 29) had labial herpes. All of them had 1 rectal suppository consisting of 4 mg active substance before sleep on the 1 st , 2 nd , 6 th , 10 th and 14 th days. Circulating interferon was detected in sera of 23 patients before therapy and on the 7 th day during the 1 st course. Circulating interferon was detected by the biological standart micromethod.Results. 30 of 36 patients ended the therapy completely. 6 discontinued therapy of various reasons. None of them discontinued therapy because of side effects of Larifan. 66% (n = 20) patients had no relapses during therapy, 17% (n = 5) patients had 1 relapse, 3% (n = 1) had 2 relapses, 7% (n = 2) had 3 relapses, 7% (n = 2) had 5 relapses.The increase of circulating interferon was detected in 87% (n = 16) patients, only 13% (n = 7) of patients it remained low.Conclusion. The results prove that a suppository variety of Larifan combines both the local and systemic immune modeling properties. Larifan would be an efficient medicine to treat frequently recurring herpes infections during their aggravations. To evaluate the significance of Larifan suppositories in the treatment of frequently recurring herpes infections further long term researches are needed and are carried out. Multidimensional Liquid Phase Separations of Intact Proteins as an Alternative to 2D Gel Electrophoresis for Proteomics. A. Apffel, 1 A. Adler, 1 T. Sana, 1 J. Garcia, 1 R. Kincaid, 1 S. Udiavar. 1 1 Molecular Technologies Laboratory, Agilent Laboratories, Palo Alto, CA, USA.In the field of expression proteomics, the dominant separation technology has been Two Dimensional Gel Electrophoresis (2DGE) frequently coupled with Mass Spectrometric identification methods. Although an extremely powerful separation technique, 2DGE suffers from a number of drawbacks including lack of speed and automation, poor reproducibility and quantitation and fundamental limitations in linearity resulting in a bias towards highly expressed proteins. Alternative approaches based on Multidimensional HPLC separation of proteolytic digest of complex samples have emerged combining nanoscale reversed phase separations of strong cation exchange fractions. Although these approaches have demonstrated utility, the complexity of the resulting mixture represents a considerable chromatographic challenge. Furthermore, pooling the proteolytic fragments from a large number of proteins eliminates any association of a given peptide with its parent proteins and thus configurations of multiple Post Translational Modifications.In an effort to overcome the limitations of existing techniques, we have been evaluating the use of multidimensional liquid phase separation of intact proteins as an alternative. In this approach, 96 fractions from a first dimension are collected and re-fractionated by a second, orthogonal separation mechanism collecting 16 fractions resulting in a total of 1536 fraction per sample. As 1 st dimension modes, we have coupled strong anion exchange chromatography with a 2 nd dimension based on high speed separations using a 5mm 3002 Macroporous Reversed Phase material at high temperatures (608C) for very high resolution and recovery. This material allows rapid diffusion of large molecular proteins resulting reversed phase cycle times of 5-10 minutes/fraction. The combinations of these chromatographic modes are largely orthogonal, utilize compatible mobile phases and yield resolution of several hundred proteins in a single analysis.Following the 2D Fractionation of intact proteins, all of the collected fractions are reduced, alkylated and proteolytically digested for subsequent analysis by Nanoscale electrospray LC/ MS based on a microfluidic ChipMS System for Ion Trap Mass Spectrometry. Although each fraction generally contains multiple proteins, it is feasible to identify the components using rapid Nanospray LC-MS/MS information. Following collection of the MS/MS data, the pooled spectra are processed and compared to protein library data bases using SpectrumMill software to identify proteins present in the samples.This method is demonstrated with the analysis of a protein extract of Murine Immunodepleted Sera from NOD and NOD.B10 Mice. Oligosaccharides are involved in a host of cell-cell processes and play a key role in the immune system. Changes in glycosylation or the formation of aberrant oligosaccharides accompany a host of diseases from infection to cancer (eg. ovarian cancer, diabetes, cystic fibrosis, arthritis, autoimmunity, respiratory capacity diseases). In this presentation, we introduce a new fully automated and integrated analytical platform for oligosaccharide profiling consisting of a chip-based chromatography system in conjunction with time-of-flight mass spectrometry. The microfluidic HPLC chips are made of laser ablated and laminated biocompatible polyimide films. High resolution separations of oligosaccharides are achieved with porous graphitized carbon (PGC) column material packed in the chip device. Oligosaccharide isomers are readily separated and resolved. In this way, over 100 different oligosaccharides are simultaneously identified and monitored. Those known to have immunostimulatory effects were unambiguously identified. This new advanced chip-based analytical platform is also capable to analyze other aspects related to diseases like changes in the phosphorylation pattern or changes at the metabolomics level. Ischemia/reperfusion (I/R) injury consists of an acute inflammatory response involving complement which is activated upon the recognition of natural IgM to self-antigen exposed by ischemia. This study dissected the role of lectin versus classical complement pathways in an intestinal I/R model. When RAG-1-/-mice were reconstituted with I/R-specific clonal IgM, mannose-binding lectin (MBL) co-localized with IgM in the injured tissue. Further, MBL-/-mice were protected from I/R injury (40min ischemia, 3hour reperfusion) with the absence of IgM and C3 deposition. In contrast, C1q-/-mice were injured similarly as WT animals with deposition of MBL and IgM in the damaged tissue. Reperfusion in MBL-/-mice for 15min showed IgM deposition in intestinal villi, and subsequent proteomic analysis identified specific self-antigen bound to IgM. This study reveals that I/R pathogenesis is initiated by IgM response to ischemic antigen followed by direct activation of downstream lectin pathway of complement. Effect of Plaferon LB on the Damaged Peripheral Nerve Regeneration. T. Chikovani, 1,2 M. Kvezereli, 2 G. Burkadze, 1 T. Giorgadze, 2 V. Bakhutashvili. 2 1 Microbilogy, Virology & Immunology, Tbilisi State Medical University, Tbilisi, Georgia; 2 Biomedicine, Institute of Medical Biotechnology, Tbilisi, Georgia.Axonal repair of mature neurons involves complex molecular changes. Critical role is played by Schwann cells as well as macrophages and inflammatory cells. One of the most important molecules, which determine degeneration/regeneration processes in damaged nerve, is nitric oxide (NO). NO influences on many aspects of nervous system physiology, being either detrimental or beneficial. As it is already revealed, the possible NO targets are neurofilaments and myelin sheaths of interrupted axon, newly formed neuromuscular endplates and Schwann cells in the distal nerve stump.Manipulation of NO supply may offer interesting therapeutic options for peripheral nerve lesions recovery. The possibility of regeneration of transected sciatic nerve axon under the influence of Plaferon LB (PLB), inhibiting inducible nitric oxide synthase activity in vitro was investigated.Experiment was conducted on twenty white rats. Animals were daily treated by PLB or saline. Ten animals were treated with PLB, others were undergone a course of saline treatment. Operation was carried out by microsurgeon; sciatic nerve was transected and than sewed up.Animals were sacrificed seven and thirty days after operation. The nerve was removed for nitric oxide (NO) measurement (after 7 days) and morphological examination (after month). NO was measured by electron spin resonance method using NO trap.For morphological investigation, preparations were stained by hematoxylin-eosin methods, neurohistological method-Nils and immunocytochemical method by using monoclonal antibody S100.Our findings suggest that PLB may play an important role in the regeneration of the injured peripheral nerve. In the injured nerve intense increase of NO production as well as the quantity of Shwann and mast cells was observed. According to our results PLB prevents the induction of NO production in axotomized sciatic nerve. Quantitative analysis showed that under the influence of PLB increase of Shwann and mast cells was statistically high reliable. IgA nephropathy (IgAN) is characterized by glomerular codeposition of IgA and complement components. The complement system can be activated via three activation pathways, the classical pathway, the alternative pathway, and the more recently identified lectin pathway. IgA can activate the complement system via the alternative pathway but not the classical pathway. Furthermore, recent data indicate involvement of the lectin pathway of complement in IgAN. The LP can be activated by binding of mannose-binding lectin (MBL), L-ficolin and H-ficolin to carbohydrate ligands, followed by activation of MBL-associated serine proteases (MASPs) and C4. We studied the potential role of the lectin pathway in IgAN and the interaction between MBL and human IgA as a possible explanation for lectin pathway activation in IgAN.Renal biopsies of IgAN patients (n = 60) were stained for IgA1, IgA2, MBL, L-ficolin and complement. Polymeric and monomeric serum IgA of IgAN patients (n = 14) and healthy controls (n = 8) was purified by affinity chromatography and gel filtration. Glomerular deposition of MBL was observed in 15 out of 41 cases with IgAN (25 %) and showed a mesangial pattern. All MBL-positive cases, but none of the MBL-negative cases, also showed glomerular deposition of L-ficolin, MASP-1/3, C4-binding protein and C4d. Glomerular deposition of MBL and L-ficolin was associated with significantly more mesangial proliferation, interstitial damage, and proteinuria.In vitro studies demonstrated binding of MBL to IgA from healthy controls and from IgAN patients, with a strong inter-individual variation. MBL binding was observed to polymeric but not to monomeric IgA. The interaction between MBL and IgA was dose-dependent and could be inhibited by EDTA and D-mannose but not L-mannose, indicating involvement of the lectin domain of MBL.Together, these findings strongly point to a role of the lectin pathway of complement in glomerular activation of C4 in IgAN, and suggest a contribution for both MBL and L-ficolin in the progression of the disease. Furthermore, the findings support the hypothesis that glomerular MBL binding and complement activation in IgAN is based on an interaction of MBL with IgA.F2.29. Characterisation of Antibody Titres in an Intravenous Immunoglobulin Concentrate (FlebogammaR).M. Lopez, 1 J. I. Jorquera. 1 1 R&D Area, Instituto Grifols, S.A., Parets del Valles, Barcelona, Spain.To evaluate the IgG antibody levels in an intravenous immunoglobulin concentrate (FlebogammaR) against several pathogens.From 3 to 217 lots of FlebogammaR, product at 5% IgG concentration containing 5% sorbitol as excipient, were studied. The titres against Cytomegalovirus (CMV), hepatitis A and B, measles, varicella (VZV), parvovirus B19, poliovirus type I, II and III, rubella, Epstein Barr (EBV), herpes simplex type 1 and 2, influenza A and B, parainfluenza type 1 and 2, adenovirus, mumps, coxsackie, echovirus, tetanus, diphtheria, streptococcus pneumoniae, candida albicans, bordetella pertussis, helicobacter pylori, campylobacter jejuni, chlamydia, borrelia burgdorferi and toxoplasma gondii, were studied. ELISA tests were used except for antibodies to poliovirus type I, II and III, diphtheria and measles, which were performed by neutralization tests. The results are shown as IU/ml or Elisa Units (U/ml) when no international reference is available. For determination against poliovirus, measles and diphtheria, the CBER lot 176 and US Diphtheria antitoxin standard F4505 reference preparations were used.The most relevant results obtained are as follows: hepatitis A and B (35 F 4 and 1.1 F 0.5 IU/ml respectively), VZV (12 F 1 IU/ ml), tetanus (22 F 4 IU/ml), parvovirus B19 (141 F 21 IU/ml), CMV (31 F 6 IU/ml). For all these agents, the titres in the final product are 6-to 9-fold the value observed in the starting plasma. The results obtained for neutralizing antibodies against poliovirus type I, II and III and measles were 0.34 F 0.11, 0.71 F 0.13, 0.59 F 0.10 and 0.30 F 0.08 sample/reference ratio, respectively, and against diphtheria 4.4 F 0.5 U/ml. With regard to the other antibodies studied, since no international reference is available, the quantitation is performed in relation to the cut-off or positivity limit of the ELISA method used. Thus, the activity against EBV (644 F 50 U/ml), herpes (9290 F 735 U/ml), mumps (1890 F 169 U/ml), helycobacter (436 F 41 U/ml), adenovirus (62 F 9 U/ml), rubella (323 F 37 U/ml), St. pneumoniae (449 F 57 mg/L), influenza, etc., is 5-fold the cut-off limit. Finally, antibodies are also detected, even though to a lower extent, against Candida (50 F 5 U/ml), Bordetella (28 F 2 U/ml), Campylobacter (90 U/ml), Coxiella (42 F 21 U/ml) and Coxsackie (220 F 11 U/ml).FlebogammaR contains IgG antibodies against a wide panel of pathogens, between 6-and 9-fold the normal plasma values for a high number of agents. H. Biescas, 1 R. Gajardo, 1 A. Vandermeeren, 2 M. Esteban, 2 J. I. Jorquera. 1 1 Research and Development Area, Instituto Grifols, Barcelona, Spain; 2 Centro Nacional de Biotecnologia, CSIC, Campus Universidad Autonoma, Madrid, Spain.Immunocompromised individuals may be at risk for Vaccinia infection if widespread smallpox-immunization programs are needed. The aim of this study was to determine the level of neutralizing antibodies to Vaccinia virus in Human Intravenous Immunoglobulin (IVIG) manufactured by Instituto Grifols. Thirteen batches of FlebogammaR were evaluated for the presence of anti-Vaccinia antibodies by a Vaccinia Plaque reduction neutralization assay. The Vaccinia virus working stock (Western Reserve-WR strain) was mixed with IVIG preparations, and after incubation at 378C for 1h were inoculated onto monkey cells (BSC-40) for detection. An international standard from NIBSC (63/024) (potency of 1000 IU/ml) was used as the Antibody-positive Control preparation. Sera from non-smallpox vaccinated donors was the negative control preparation used. There is a linear correlation between the rate of virus neutralization and the antibody concentration. The neutralizing titer for a specific sample was expressed as the sample dilution that reduced the number of virus plaques to 50% (determined in triplicate) as compared with the number seen in the virus positive control (without IVIG added).The results of the experiments provided a neutralizing potency of 35 (range 18 to 68) anti-Vaccinia IU/ml IVIG. Anti-Vaccinia antibodies in the product were also determined by ELISA and WB techniques revealing that the product was able to react against several Vaccinia virus proteins. There were no relevant differences among batches in the anti-Vaccinia titers.The results of this study are consistent with the work of Goldsmith et al. The titers of anti-Vaccinia antibodies found in this preparation have biological relevance since they provided protection against Vaccinia infection to immunocompromised mice. Compatibility Study of Two Intravenous Immunoglobulin Preparations with Plastic Containers. M. Lopez, 1 M. Costa, 1 J. I. Jorquera. 1 1 R&D Area, Instituto Grifols, S.A., Parets del Valles, Barcelona, Spain.The customization of intravenous immunoglobulins (IVIG) therapeutical doses to each patientTs needs, by unifying the content of several bottles in a single container, is more and more frequent.This study deals with the compatibility of two IVIG preparations sharing formulation (5% sorbitol) with two types of plastic container.Nine lots of two preparations manufactured by Grifols, FlebogammaR (licensed) and 5% IGIV3I (under clinical trial), both containing 5% sorbitol as excipient, were filled in Griflexpolypropylene (PP) or Gribag-PVC sterile bags, at a rate of 40 ml solution/100 ml bag, what implies worst case conditions as refers to product-plastic contact ratio. The GrifillR system, which ensures sterile filling, was used. The product, filled in the plastic containers, was stored at 5 8C, 30 8C and 40 8C for a period between 10 and 15 days. For each temperature, tests before and after transfer from the original container (glass bottle) to the plastic container (PVC or PP), as well as at different storage time points, were scheduled. The following parameters were evaluated: appearance, pH, turbidity, osmolality, total protein (Bio-Rad), molecular distribution (HPLC), anticomplementary activity (ACA), prekallikrein activator (PKA), antibodies titration against tetanus and hepatitis B (ELISA), antibodies against poliovirus type I (neutralization), DEHP (gas chromatography, mass spectrometry) and other polypropylene plastic additives: BHT, Irganox 1010 and 1073 and Ethanox 330 (HPLC), sorbitol (HPLC), pyrogens (injection into rabbits), toxicity (intraperitoneal injection into guinea pigs and mice), sterility (Steritest system, from Millipore) and bags weight control.The results of the tests performed do not show significant variations in the productsT characteristics after transferring them to the plastic containers. The results at temperatures of 5 8C and 30 8C do not show any sign of incompatibility with the plastic material, the antibody titres studied remaining perfectly stable. The markers used to assess the possible migration of plastic components to the product (DEHP for PVC) and PP additives are undetectable or very low (the DEHP content shows values below 5 Ag/ml in all instances and the PP markers are undetectable). The mean weight loss is between 0.1% and 0.2% after 10-15 days of storage at 5 8C and between 0.6% and 1.3% at 30 8C. In the accelerated temperature studies (40 8C) a higher weight loss is noticed (between 1.2% and 2.9%), but no significant variations are detected in the other parameters studied.5% sorbitol-formulated IVIG shows compatibility with PVC and PP during short time periods, between 2 8C and 30 8C. Introduction: Autoimmune hepatitis (AIH) is a rare disease characterized by progressive necro-inflammatory hepatocyte injury caused by breakdown of immune tolerance to self-antigens however pathogenic mechanisms underlying the development of AIH are still unclear.The aim of our study was a complex analysis of autoimmune phenomena involving activation and apoptosis as well as transfer of co-stimulatory signals of particular lymphocytic subpopulations in peripheral blood of patients with AIH in different stages of immunosuppressive therapy.Material and methods: Examinations were carried out in 26 AIH patients divided into two groups. The first group consisted of 13 patients with de-novo diagnosed AIH in which immunophenotyping of peripheral blood lymphocytes was done twice i.e., before and after five months of immunosuppressive therapy. The second group consisted of 13 patients showing clinical and laboratory features of AIH remission (single immunophenotyping).The functional state of lymphocytes was examined using threecolor flow cytometry technique with 17 monoclonal antibodies repertoire. The reference ranges for activation and apoptosis markers were obtained from immunophenotyping of 30 healthy volunteers.Results: The percentage of T cells, Tc with surface marker CD95 was significantly lower in AIH de-novo diagnosed patients than in healthy subjects. The percentage of activated Th, as well as B cells were significantly higher in AIH de-novo diagnosed than in healthy subjects.After five months of immunosuppressive therapy following changes with comparison to initial data were found: a) increase of percentages of T, Th and Tc cells, b) increase of percentage of Th cells with surface CD95, c) decrease of percentage of NK cells, d) decrease of percentage of activated B and B1a cells.After 2 to 4 years of immunosuppressive therapy no significant differences in percentages of T, Th, Tc and NK cells were found between AIH patients and healthy subjects.Conclusions: 1. The activated T lymphocytes appear during clinical exacerbation of AIH. The percentage of peripheral blood NK cells decreases after 5 months of immunosuppressive treatment and normalizes after 2-4 years of treatment. The percentage of activated B cells was increased in active phase of disease and significantly decreased following 5 months and 2-4 years of immunosuppressive therapy. Exacerbation of AIH is associated with a decrease number of activated Tc lymphocytes which probably results from accumulation of these cells in the liver. Activation of lymphocytes T via CD69 antigen seems to be a principal immune event in pathogenesis of AIH exacerbation. Immunosuppressive therapy lasting 2-4 years leads to normalization of amount of basic T lymphocyte populations in peripheral blood. No such effect was observed after 5 months of immunosuppressive therapy.F2.33. Identifying Common bInnate SignatureQ from Gene Expression Profile in Innate vs. Adaptive Lymphocytes.T. 1 1 Section on Immunology and Immunogenetics, Joslin Diabetes Center, Boston, MA, USA. Innate and adaptive immune responses are two radically different strategies the host immune system takes against pathogen assaults. This dichotomous view of the immune response has now been widely accepted. However, it is not clear what the definition, or hallmark, of innate immunity is, and what really distinguishes it from adaptive immunity. In order to address this question, we took a broad approach and compared gene-expression profiles in innate vs. adaptive immune cell populations. The players in innate immune responses include neutrophils, macrophages, natural killer (NK) cells, and dendritic cells (DCs), whereas those in adaptive responses include the classical T and B cells. However, comparing two extreme cell-types (e.g. neutrophils vs. T cells) could end up highlighting superficial differences such as TCR and downstream genes. Therefore, we have explored differences in gene expression by comparing close, but distinct, paired innate vs. adaptive populations; CD3 + CD4 + NK1.1 + T cells (NKT) vs. CD3 + CD4 + NK1.1-T cells (CD4T); IgM hi IgD int CD11b + B cells (B1) vs. IgM int IgD hi CD11b-B cells (B2); TCRb + CD8a + CD8b-cells (CD8aa) vs. TCRb + CD8a + CD8b + cells (CD8ab). These six populations were sorted from wild-type C57BL/6 mice or TCR transgenic mice, and mRNA was hybridized to Affymetrix U74Av2 chips. The genomic analyses were done in two ways: a mathematical/geometrical analysis of the cell population coordinates based on their gene expressions (Reference Population Plot; RPP), and a bioinformatic analysis of the over-expressed genes based on their molecular functions. The RPPs indicate that innate lymphocytes have active-, memory-, NK-, and regulatory-like characters. We found 22 genes over-expressed N1.5 fold in the intersection of three innate vs. adaptive comparisons, a number which is significantly higher than the background level (1 gene) in randomized datasets. We also defined 33 genes that are under-expressed in innate cells. These two sets of genes successfully sort multiple lymphocyte populations into innate and adaptive subgroups in the RPP, indicating that expression levels of the 22 genes can be designated as an bunbiasedQ innate signature. The molecular characterization of the genes induced in innate populations pointed to multiple molecular pathways being activated. We have found a limited set of functional entities to be enhanced across the populations of innate lymphocytes: essentially the intracellular vacuole trafficking and interferon-inducible GTPase systems. This finding prompts one to speculate that the innate immune system might deploy a unique intracellular recognition system to detect subtle changes in the tissue environment. Our analyses have revealed shared genes and pathways induced in innate lymphocytes, and thus a common binnate signatureQ that distinguishes innate lymphocytes from adaptive ones. These findings are not only instrumental for genomic definition and classification of binnatenessQ in various immune cell-types, but also have implications for our understanding of the origin and evolution of innate immunity. Previously, we reported that the saccharidic structure (Gal, GalNAc) recognized by Amaranthus leucocarpus lectin (ALL) is expressed on activated T cells. The objective of this work is to determine if the structure recognized by ALL is similar to the structure recognized by PNA.PBMC were obtained form healthy donors and cultured in presence of 1 mg/ml of Concanavalin A (Con A). Then, the cells were recovered every 24 h during 4 days. After that, the cells were labeled with the FITC conjugated-lectins Amaranthus leucocarpus (ALL) or Peneaut agglutinin (PNA) and against different mAb such as CD3-Cychrome, CD69-PE or CD25-PE. In some cases the cells were treated by neuraminidase to eliminate the sialic acid camouflage. The results were analyzed by flow cytometry.Results: After stimulation with Con A, the percentage of ALL+ T cells increases from 3% to 69% at 48 h, diminishing gradually, whereas the percentage of cells PNA+ T cells increases from 1% to 90% at 72 h and maintaining this percentage until 96 h. The expression of CD69 and CD25 and we observed that the CD69 and CD25 expression was 3 times more on ALL+ T cells. However on the neuraminidase treated cells the expression of CD69 and CD25 was disminished in both, ALL+ and PNA+ T cells.Conclusions: Our results suggest that saccharidic structure recongnized by ALL is different to the saccharidic structure recognized by PNA. Short-Term Atorvastatin Treatment Enhances Specific Antibody Production Following Tetanus Toxoid Vaccination in Healthy Volunteers. Statins are a class of HMG CoA reductase inhibitors used by millions of Americans with high cholesterol. Several lines of evidence suggest that these drugs possess anti-inflammatory and immunomodulatory properties beyond their cholesterol-lowering effects. Recent reports have shown that statins affect immune responsiveness by skewing the T H 1/ T H 2 balance and interfering with MHC class II expression. While inhibition of inflammatory processes can be protective, suppression of immune responses such as antigen presentation may weaken host defense. To determine whether HMG CoA reductase inhibitor treatment has an effect on in vivo acute phase response and antibody production following tetanus vaccination in normal healthy volunteers in a double blind, parallel, placebo controlled study. Twenty healthy volunteers were randomized to receive atorvastatin 40 mg (AT) or calcium placebo (PL) for 10 days and all volunteers received tetanus toxoid (TT) vaccine on day 5 of the study. The acute phase response was evaluated by determining changes in ESR, CBC with differentials, and serum concentrations of acute phase proteins (a 1 -antitrypsin, C-reactive protein, and a1-acid glycoprotein) and cytokines (IFN g, IL-4, IL-6, and IL-10). The humoral response to vaccination was assessed by measuring total immunoglobulins and specific anti-TT antibodies. Baseline measurements of all variables were similar in both groups. Following the ten-days of study drug or placebo, subjects in the atorvastatin group had a significant reduction in total cholesterol (AT: À44.4 F 9 mg/dL; PL: 22.7 F 8 mg/dL; P b 0.0001) and LDL cholesterol (AT: À46.2 F 9 mg/dl; PL: 13.2 F 7 mg/dl; P = 0.0001) compared to the control group, demonstrating the effectiveness of atorvastatin. The baseline anti-TT antibody concentrations and the number of years elapsed since last vaccination were similar in both groups. Unexpectedly, the production of specific antibody against TT (predominately IgG 1 ) was more than 3 fold higher in volunteers treated with atorvastatin 15 days post-vaccination (AT: 2306 F 468 units; PL: 713 F 21 units, P = 0.0085). Atorvastatin suppressed the post-vaccination rise in platelet counts (AT: À5.2 F 3.1%; PL: 9.63 F 4.4%; P = 0.0132) and lymphocyte counts (AT: À7.4 F 9.1%; PL: 33.4 F 10.5%; P = 0.0089) consistent with its anti-inflammatory properties. There were no significant changes in ESR, serum levels of acute phase reactants or cytokines in the treatment and control groups. This study demonstrates that statins augment specific immune responses following vaccination in healthy volunteers rather than suppress them. Beyond their applications in reducing cholesterol and suppressing inflammation, it is possible that statins may benefit groups who respond poorly to vaccinations, such as the elderly or immunocompromised patients. Further clinical studies are needed to determine if these groups demonstrate a similar response to atorvastatin as well as to determine the mechanism(s) that mediated this effect. Osmel Gaspar Guerra Segura. Dr. A. Neto, Guantanamo, Guantanamo, Cuba.We did a pedagogical diagnostic of the knowledges of Immunology to the students of second year of Medicine before and after the teaching the topics in Microbiology and Pathology, then the diagnostic was done to the six year students and Internal Medicine residents. To accomplish the objetives were designed some pedagogical instruments (interview, questions, TestTs knowledges) to students and proffesors wich taught and recieved the subject. The 93 % of the knowledges were adquired by the second year students, and the Internal Medicine residents obtained the lowest marks. The highest integration of contents of Immunology with other subjects is only about 40 %. ThatTs right we propose an Immunology syllabus in agreement with social and professional needs of cuban doctors and from other countries. C1 inhibitor deficiency is a rare condition that can be either inherited (hereditary angioedema, HAE) or acquired (acquired angioedema, AAE) and is clinically characterized by recurrent, selflimiting skin and intestinal edema and life-threatening upper airway obstruction. Replacement therapy with C1-INH concentrate is the treatment of choice for severe acute attacks of angioedema in Europe.We report the need and efficacy of C1-INH concentrate in 477 patients with HAE and 25 with AAE observed for: 30-25 years 50 patients, 24-20 in 44, 19-15 in 48, 14-10 in 96, 9-5 105, b5 in 159. Side effects were recorded. One HAE patient underwent tracheostomy for laryngeal edema despite treatment with C1-INH concentrate. In 45 peripheral attacks the time to resolution was within one hour in 23 episodes (51 %), between 1 and 2 hours in 8 episodes and between 2 and 48 hours in 14 episodes. In 5 patients with AAE (54 infusions) beginning of resolution was always within one hour; in the remaining 3 patients (35 infusions) the response became progressively slow 3 (3 hours or more) requiring higher doses of C1-INH concentrate. Two anaphylactoid reactions were reported in 2 HAE patients. No HIV infection was ever detected. Our data indicate that treatment with C1-INH concentrate is highly effective in angioedema of the laryngeal or abdominal mucosa; its effectiveness is reduced when the skin is involved and particularly in peripheral attacks. Safety is generally good and transmission of blood borne infection has drastically reduced since viral inactivation procedures have been introduced. Immunonutritional Assessment of Special PlasmaTs Donors. Osmel Gaspar Guerra Segura, Nerys Noa Rdguez. Dr. A.Neto, Guantanamo, Guantanamo, Cuba; 2 Quality Control, Provincial Blood Bank, Guantanamo, Guantanamo, Cuba.We did a prospective study of special plasmaTs donors with more than a year in the plasmapheresis program of hiperimmune anti TT (tetanic toxoid) plasma in the provincial Blood Bank of Guantanamo. We have evaluated the donors nutritionaly with the CERES program from Hygene of Foods Institute from Hanvana wich have an special form for the collection of data in the relation to high, weight and race. From the immunohematologic and biochemist point of view, we value each fractions of Protein Electrophoresis, Eritro, Hemoglobin and in particular the specific antibodies (anti TT) and positiveness to antigens of Hepatitis B and C and HIV test. From the nutritional point of view the lipids supply is the only deficiency in those donators where antropometric and hematologic parameters are agree with literature. Presentation of the Human Hepatitis B Surface Antigen-Loop by the Cpn10 Scaffold Induces a Specific Antibody Response in Mice by Genetic Immunization.S. Neckermann, 1 J. Lohrmann, 2 W. Zimmermann. 1 1 Tumor Immunology Laboratory, Department of Urology, University Clinic Grosshadern, Ludwig-Maximilians-University, Munich, Germany; 2 GENOVAC GmbH, Freiburg, Germany. Introduction: Cpn10 proteins belong to a widely distributed protein family found from mammals to bacteria. Because of their conservation between species they exhibit low immunogenicity. This property is advantageous when fusion proteins are used in genetic immunizations: antibodies against the fusion partner and not against Cpn10 are generated. This native loop can be replaced by a foreign loop sequence.An important application of this bloop replacementQ strategy lies in presenting extracellular loops of transmembrane proteins like G-protein coupled receptors (GPCR) for which it has been proved difficult to generate antisera against their short extracellular loops. The broad interest on the GPCR family is based upon the fact that 50-60% of approved drugs elicit their therapeutic effect by selectively addressing GPCRs.As a proof of principle for the applicability of the Cpn10 scaffold we have replaced the Cpn10 loop by the baQ determinant of the Hepatitis B surface antigen (HBsAg). The baQ determinant is a small loop of nine amino acids, which is highly immunogenic when presented in the context of the full length HBsAg. In this study, we wanted to analyze whether presentation of this HBsAg loop in the Cpn10 scaffold can elicit a specific immune response upon genetic immunization and whether the scaffold is able to mimic the native conformation. Therefore we immunized mice with a plasmid coding for the Cpn10-HbsAg scaffold and analyzed the antisera for specific anti-HBsAg antibodies.Methods:The murine Cpn10 cDNA and the nucleotides coding for the baQ determinant were cloned into an optimized immunization vector. BALB/c mice were immunized once in a two week interval over a period of 12 weeks using a HeliosR gene gun. Blood samples were taken from each animal every two weeks and analysed for the presence of specific antibodies using a cell-based ELISA assay.Results: Screening the antisera in the cell-based ELISA showed that all mice elicited specific antibodies against the plasmid-encoded Cpn10-HBsAg antigen. It could also be demonstrated that this immune response was not directed against the scaffold itself proving the low immunogenicity of the scaffold in the murine system. Therefore the detected antibody response seems to be specific for the baQ determinant of the HBsAg.Discussion/Conclusion: We could show for the first time that the murine Cpn10 scaffold can be used in genetic immunization experiments for presenting a foreign peptide loop and for eliciting an immune response against this encoded antigen-loop. Whether this scaffold allows formation of the native loop conformation needs to be further investigated in an ELISA, where the recombinant HBsAg protein should be detectable by the sera. Futher experiments have to be performed to establish whether this approach can be extended and used e.g. for the directed generation of antibodies against GPCR loops. Angioedema without major urticaria flares is poorly characterized, it is caused by different conditions and few data exist on the underlying etiopathogenetic mechanisms. Here we report our experience on patients with this symptoms and propose a diagnostic-therapeutic approach.Methods 929 consecutive patients were examined at our outpatient clinic for recurrent angioedema without urticaria between 1993 and 2002. Known causes of angioedema were identified by clinical history with detailed information about personal and familial allergies; relationship of angioedema to potential causative agents were search (food, drugs, insect stings); complete physical examination, routine laboratory tests (blood cell count, protein electrophoresis, erythrosedimentation rate, stool for ova and parasites, pharyngeal and urine cultures, sinus and dental film, anti-tissue autoantibodies, rheumatoid factor), complement parameters (C1 inhibitor, C4, C1q) were performed. Further testing was done when pertinent based on clinical finding. When all was negative, response to H1antihistamine was considered.Results According to our testing angioedema were classified as follow: related to external agents (drugs, insect stings, food) 209 (22.5%) (in particular 85 of them were related to treatment with an ACEinhibitor); associated to autoimmune diseases or infections 77 (8.2%); hereditary C1 inhibitor deficiency 183 (19.6%); acquired C1 inhibitor deficiency 14 (1.5%); idiopathic histaminergic 253 (27.2%), idiopathic non histaminergic 40 (4%). Conclusion Based on the frequency of the different diagnosis we propose the following progression of testing: 1. anamnestic identification of causative agents, 2. testing for complement abnormalities, 3. H1 antagonist treatment, 4. complete diagnostic workup. T cell activation by intestinal dendritic cells (DC) induces guttropism. We show that, reciprocally, DC from peripheral lymph nodes (PLN-DC) induce homing receptors promoting CD8 T cell accumulation in inflamed skin, particularly ligands for P-and Eselectin. Differential imprinting of tissue-tropism was independent of Th1/Th2 cytokines and not restricted to particular DC subsets. Fixed PLN-DC retained the capacity to induce selectin ligands on T cells, which was suppressed by addition of live intestinal DC. By contrast, fixed intestinal DC failed to promote gut-tropism and instead induced skin-homing receptors. Moreover, the induction of selectin ligands driven by antigen-pulsed PLN-DC could be suppressed din transT by adding live intestinal DC, but PLN-DC did not suppress gut homing receptors induced by intestinal DC. Reactivation of tissue-committed memory cells modified their tissue-tropism according to the last activating DCTs origin. Thus, CD8 T cells activated by DC acquire selectin ligands by default unless they encounter fixation-sensitive signal(s) for gut-tropism from intestinal DC. Memory T cells remain responsive to these signals allowing for dynamic migratory reprogramming by skin-and gut-associated DC. ( A Prospective study has been carried out to study the bacteriocin producing abilility of water isolates a total of 27 isolates were screened for their bacteriocinogenic potential against intragenic and intergenic microorganisms 20% of the isolates were found to exert broad range bacteriocinogenesis (bioactivity). However none of the producers was antagonistic against the related streptococcal strains.Bacteriocinogenesis was demonstrated by 1) stab over lay 2) cross streak 3) patch test4) Agar well.Titration of bacteriocine was done by arbitary unit(AU) method.In First method test strains were stabbed into Luria agar plate and incubated at 378C for 24 hrs, chloroform was used to kill the bacteria, plates were than overlaid by soft agar (0.6%) containing 5-10 micro liter of 2-3 hours grown indicator strainsand incubated at 378C for 24 hrs and examined for zone of inhibition In second Test strains were streaked across the surface of a Luria agar plate and incubated at 378C over night the growth of test strain was removed and plate was exposed to chloroform as described earlier, indicator strain was then cross streaked.Plate was incubated at 378C over night,and examined for inhibition of growth.In 3rdA luria agar plate was overlaid with 3 ml soft agar containing 5-10 micro liter of indicator strain. A fresh colony of strain was stabbed into indicator lawn.Plate was incubated at 378C for 24 hrs in 4th Test strain was grown in Luria broth,culture was centrifuged at 3000 rpm for 20 min, supernatant was collected. Luria agar plate was overlaid with 3 ml soft agar containing 5-10 micro liter indicator strain. Wells were made and 100-200 micro liter supernate was added in the well. For titration, Inoculate the nutrient broth by different producers strains and incubated at 378C, next day centrifuge those tubes for 20 minutes then make 2 fold dilutions (1:2-1:4,so on)on nutrient agar make 5 wells and marked according to dilution tubes (1:2-1:32).Take 50 micro liter from 1:2 dilution tube and transfer in 1:2 marked well and this processwas repeated with all dilutions. Next day check the zone of inhibition.Among many results most appropriate results obtained about Listeria monicylogenes and Strepptococcus Pyogenes Bacteriocinogenic organisms gave this activity against some Gram negative stains such as E.coli, S.typhi, Klebsiella, Pseudomanas and some G+ve stains such as S.aureus, S.fecalis, M.leutus, Bsubtilus. Streptococcin titer was estimated to be 1:64 A.U. The lacuna percentage (which determine the number of Streptococcin producing clones in a population) was found to be 14.6 elevated temperature 408C mediated curing of the producing isolates revealed the chromosomal location of bacteriocinogenic determinantsas reported in the past studies. Cardiac surgery is usually followed by adhesions, which still represents an incompletely solved problem. Different treatments have been proposed, the fibrin seal probably been the best so far, reducing adhesions by about 50%. Entamoeba histolytica produces an anti-inflammatory peptide (Met-Gln-Cys-Asn-Ser) termed Monocyte Locomotion Inhibitory Factor (MLIF) for its first described effect. Monocytes are key cells of the late inflammation and also modulates the cicatrization stage of the wound-healing process. The purpose of this study is to prevent the formation of experimentally induced pericardic adhesions in rats (a model originally developed to induce cardiac colateral circulation). Epicardiectomy and 3x soft sandpaper scratches over 0.5 cm 2 apical area of the heart were performed in four groups of eight male Wistar rats each under anesthesia and sterile conditions. Groups received either; MLIF 100 Ag in 0,05 ml of pyrogen free saline solution, fibrin seal solution (0.1 ml), both, or none, the final volume adjusted to 0.1 ml with saline solution. All the animals recovered and were humanly sacrificed at day ten with an anesthetic overdose. Systematized necropsies and photographic registers were done, and were blindly evaluated by three independent participants. MLIF together with fibrin seal completely inhibits the pericardic adhesions in this model (++++ to 0, pb0.05). That combined MLIF and fibrin seal inhibited adhesions but either substance alone induced only a partial-and qualitatively different-reduction of adhesions suggests that these factors have different target mechanisms and act synergically, namely MLIF reducing activation signals from monocytes to fibroblasts, while fibrin seal modulating the Pompe disease is an autosomal recessive lysosomal storage disorder characterized by the deficiency of acid alpha-glucosidase (GAA). Infantile Pompe patients experience life-threatening manifestations such as cardiomegaly and late-onset patients experience progressive myopathy with or without respiratory insufficiency. The use of recombinant human alpha-glucosidase (rhGAA) is being investigated as a therapeutic agent for Pompe disease. In our clinical studies, it has been observed that patients treated with rhGAA can seroconvert in response to the therapeutic protein. The role of these rhGAAspecific antibodies is unclear, but they could potentially inhibit the activity of the therapeutic protein or alter the biodistribution, thereby decreasing enzyme efficacy. We have previously demonstrated that weekly intraperitoneal administration of rhGAA in 6 neo /6 neo GAA knockout (GAA KO) mice results in high rhGAAspecific IgG titers. We have shown that high doses of methotrexate (MTX) were able to suppress these titers ten-fold when administered 24 and 48 hours following the first eight weekly rhGAA treatments. Further studies were conducted in GAA KO mice to both model intravenous administration of rhGAA which is the clinical route of therapeutic administration and to identify a lower efficacious dose of methotrexate. We have observed that intravenous delivery of rhGAA in GAA KO mice significantly reduces rhGAA-specific IgG titers from those observed upon intraperitoneal administration of rhGAA. In addition, we have demonstrated that MTX is most effective at reducing this immune response when administered 0, 24 and 48 hours following the first eight weekly rhGAA treatments. Moreover, lower weekly doses of MTX are as effective as the higher dose at inhibiting the rhGAAspecific immune response when given 0, 24 and 48 following rhGAA treatment. These studies suggest that the timing of MTX treatment influences its ability to inhibit the rhGAA-specific immune response. This approach may be effective in minimizing drug-specific antibody responses in patients receiving protein therapeutics. The release of metal ions from dental metal fillings is being supported by presence of galvanic cell in the oral cavity and such a way can cause local or general pathological problems in sensitive and genetically susceptible individuals.Aim of the study: To investigate the in vitro lymphocyte responses to metals before and after replacement of electro active dental metal fillings in patients with oral discomfort.Methods: Sixty-eight patients with oral discomfort and dental metal fillings were investigated. They were divided into two groups. Group A (38 persons)-patients with pathological levels of galvanic currents and group B (30 persons)-patients with physiological levels of galvanic currents and voltage. The galvanism measurement was performed by the equipment Odontologic 2000 (Embitron CZ). The lymphocyte activity was tested by the lymphocyte proliferation method modified for metals (MELISA) in 33 patients from Group A and 24 patients from group B. The electro active metal fillings replacement was performed in 18 patients from group A. Follow up MELISA at least half a year after fillings replacement was performed.Results: The higher lymphocyte activity to metals was discovered in both examined groups. The highest levels of proliferation activity were found to Ni, Hg and Mo in Group A and to Hg, Ni and Au in Group B. In general, lymphocytes of patients in group B were sensitized more than the ones in group A. The follow up results after electro active fillings removal show that lymphocyte reactivity to at least all metals tested decreased. The highest decrease was found in reaction to inorganic mercury.Conclusion: The long-time influence of galvanism can, only in sensitive and genetically susceptible individuals, influence the lymphocytes proliferation. The beneficial effect of dental metal fillings replacement was confirmed by decrease of lymphocyte reactivity to metals. Our findings support the hypothesis that suffering from the oral discomfort is not only a subjective feeling of the patient but that it can be based on a real cell discomfort due to release of metal ions from dental metal fillings and due to galvanism. But the activity of lymphocytes to metals can be influenced by a couple of other factors as well.Acknowledgment: The study is supported by the grant of Czech Ministry of Health nr. Introduction:Various un-towards reactions (Allergic/Toxic) had been observed during the course of treatment for patients with pulmonary/extra pulmonary tuberculosis.Methods: The old theme about tuberculosis as duntreatableT has been replaced by the dcurableT with introduction of the effective anti-tuberculosis drugs, but still their effectiveness is with in the limit of careful assessment of patients for ATD.Allergic & toxic manifestations are as under.Rifampicin: 1 (Urticaria,red skin with orange discoloration). 2 (Febrile reactions), 3 (Hepato), 1 (Neuro&Nephrotoxicity, blood dyscrasia haemolysis and shock, blurring of the vision, confusion, ataxia/peripheral neuritis,Pseudomembranous colitis).Ethambutol: R (Skin rashes, itching/burning sensations, anaphylactoid reactions). 1 (Nausea vomiting, abdominal pain, anorexia), 1 (head ache peripheral neuropathy, hallucination dis-orienattion, mental confusion) 2 (Visual disturbance), 1 (transient impairment of liver function) 1 (gouty arthritis).Isoniazide: R (Itching/Skin rashes, Slurred speech, hallucination,coma,generalized convulsion, status epilepticus, peripheral neuropathy, hypotension respiratory failure, severe metabolic acidosis), 1 (fever, nausea, vomiting, Hepatitis). In some rare with paralysis by interfering with calcium transport in the central nervus system.Ethionamide: 2 (Severe skin rashes, acne, alopecia, photosensitivity), 1 (nausea vomiting anorexia,diarrhea, excessive salivation, metallic taste), 2 (hepatotoxicity). 1 (Mental depression, anxiety/ psychosis, Systemic encephalopathy with pellagra-like symptoms), 1 (dizziness, drowsiness, headache, convulsion, peripheral neuropathy, tremors, paresthesias). 1 (Optic neuritis, optic atrophy, diplopia, 1 Olefactory disturbances), 1 (Deafness), R (Hypothyroidism), 1 (impotence,menorrhagia,gynaecomastia), R (hypoglycemia, hypotension), R (Thrombocytopenia, Rheumatic pains).Thiacetazone: 1 (Skin rashes, nausea, vomiting, Jaundice), 1 (giddiness, bone marrow suppression,agranulocytosis). % incidence of adverse reactions as observed R=0-1%,1=5-10%,2=N10-30%,3=N30%-80%.Results: Careful selection with Appropriate diagnosis of the infectious patients are less likely associated with adverse effects.Conclusions: Incidence of adverse effects had been relatively less frequent with first line than second line ATDs treatment. BACKGROUND: Wiskott-Aldrich syndrome protein (WASP) family members (WASP, N-WASP, WAVEs) are key molecules that regulate cytoskeletal changes in response to cell surface signals. These events are critical for various biological processes including cell migration and immune synapse formation. We recently generated chimeric mice deficient for N-WASP (NWKO) and mice double-deficient for WASP and N-WASP (DKO) employing Rag-2-deficient blastocyst complementation. NWKO chimeric mice, like WASP KO (WKO) mice, did not demonstrate any change in T cell development. In contrast, WASP/N-WASP DKO mice have a marked defect in T cell maturation associated with a block in the CD4/CD8 double-negative stage, and a consequent decrease of T cell numbers in the peripheral lymphoid organs.AIM: To elucidate the mechanism underlying the aberrant T cell development in WASP/N-WASP double-deficient mice. METHODS: We deleted N-WASP selectively in T-cells from WKO mice by using the Cre-loxP system. Lymphoid organs were processed for phenotypical analysis employing standard developmental and activation markers. Colonic samples were obtained for histological analyses and cytokine measurements.RESULTS: The lck-driven N-WASP deletion in WKO mice (creDKO) during T cell development resulted in a significant decrease in thymic cellularity. We also found increased apoptotic cell death in the creDKO animals compared to WT and WKO mice. CD69+ cells were substantially decreased in both CD4 and CD8 single-positive thymocytes from creDKO mice-which may correlate directly with the increase in apoptosis. Consequently, we found significantly decreased numbers of T cells in the spleen of creDKO mice. However, we observed that the majority of these T cells present an effector phenotype (CD44hiCD62Llo). Interestingly, creDKO mice present with colitis at an earlier onset which may be associated with increased severity.CONCLUSIONS: These studies demonstrate redundant functions for WASP and N-WASP with a critical combined role for both proteins in T cell development and function. Together the functional analyses of creDKO thymocytes suggest that the decreased migratory activity of these cells might be related to decreased signaling resulting in increased cell death and consequent reduced thymocyte numbers. Furthermore, the finding of significant increased proportion of effector T cells in the periphery of cre-DKO may be associated to the early onset of inflammatory bowel disease. FAS Induced Apoptosis-A Model System for BiFAR TM Implementation. H. Kalinski, A. Chajut, A. Khan, R. Skaliter. QuarkTs proprietary BiFAR TM platform is based on the discovery of essential functional genes using Gene Inhibitory Elements (GIEs; antisense, shRNAs or dominant negative peptides). Abundance of GIEs is expected to change based on their ability to inhibit certain genes. GIEs that are protective to a certain treatment will be enriched and GIEs sensitizing to a certain treatment will be depleted.In this study, PC3 cells were tranduced with a GIE library, propagated and then exposed to high or sub-lethal doses of anti-Fas antibody. Using a custom oligonucleotide Agilent microarray, designed to match the specific GIE library, the relative abundance of GIEs before and after anti-FAS treatment was measured. Analysis of functional GIEs identified GIEs that were protective to FAS and others that were sensitized to the treatment. Protective GIEs inhibiting genes involved in the interferon response, phosphoinositol metabolism, vesicle transport and chaperons were found. Sensitizing GIEs included those inhibiting antiapoptotic genes.The BIFAR platform, implemented on AgilentTs microarrays, allowed functional dissection of FAS induced apoptosis, a process known to participate in the killing of virally infected cells, transformed cells and peripheral auto-reactive T-cells, as well as other physiological processes. The discovery of the inhibition of genes leading to accelerated FAS induced apoptosis can be implemented in therapeutics that are aimed at the enhancement of tumor suppression or tolerance.This basic approach can be applied to a variety of other functional systems using a variety of GIE types in proliferating cells as well as fully differentiated cells, to elucidate signal transduction pathways and identify novel drug targets. Chronic brain inflammation by persistent infections, traumatisms or extensive surgeries (e.g. tumoral resection) is a main cause of secondary epilepsy. To evaluate the preservation of functional neural tissue by chronically administered antifibrotic and immunomodulating drugs in damaged brain, we induced a granuloma in the cerebral amygdala of 130 Wistar rats, by stereotaxic injection of aluminum silicate (interaural 6.2 mm, lateral 5.0 mm, height 8.5 mm, according to PaxinoTs Atlas), which were randomly distributed in 9 groups (n = 13), treated with: vehicle (water, 5% alcoholic-water or maize oil); methylprednisolone acetate (4 mg/kg/week, IM); colchicine (300 Ag/kg/ day, orally); thalidomide (160 mg/kg/day, orally); cyclosporine (10 mg/kg/day, orally); mixture of colchicine-methylprednisolone (300 Ag/kg/day-4 mg/kg/week); thalidomide-methylprednisolone (160 mg/kg/day-4 mg/kg/week); cyclosporine-methylprednisolone (10 mg/kg/day-4 mg/kg/week) during forty five days. Two weeks after the end of the treatment, experimental epilepsy was induced by PTZ subthreshold increasing doses (20-70 mg/kg), administered according 48 hours intervals, until the development of generalized seizures. Cortical electrodes were implanted in three animals from each group. Electrical activity was registered during 20 minutes. Time elapsed since the application of the drug until the first myoclonic and tonic-clonic seizure (latency), as well as the number and duration of afterdischarges were measured and compared between groups.Histological and imaging analyses and collagen quantification of granulomatous lesion were also made. Dose required to develop tonic-clonic seizures in this group was higher (P b 0.05) than that required in the rest of groups. The latency for generalized tonic-clonic seizures was also longer in thalidomide treated rats than in controls. Afterdischarges in the control group were more frequent than those in the thalidomide group, in which we also found sleep-like high voltage slow waves and isolated spikes that didnTt burst in a real afterdischarge. Thalidomide seems to preserve functional brain tissue surrounding a granulomatous lesion and would be a promissory alternative to prevent secondary epilepsy. Objective of the study: IFN-g is a pro-inflammatory effector cytokine of cell-mediated immunity that plays an essential role in both innate and adaptive phases of an immune response. Interestingly, in several Th1 dependent autoimmune models lack of IFN-g is associated with an acceleration of disease. The aim of the study was to investigate how IFN-g could be involved in the regulation of a Th1 dependent inflammation.Materials and methods: To study the influence of IFN-g on effector T cells we used an adoptive transfer model of differentiated antigen-specific Th1 cells. To differentiate the impact of IFN-g we analyzed the antigen specific delayed type hypersensitivity (DTH) reaction in different KO strains.Results: IFN-g displayed a dual function in a Th1-dependent immune reaction. In the early phase, IFN-g accelerated the inflammation, whereas in the late phase it mediated the process of self-limitation. IFN-g negatively regulates Th1 homeostasis after antigen challenge. Studies in IFN-g R KO and iNOS KO mice revealed that IFN-g could act via the receptor of host cells and that NO is involved downstream. Transfer experiments into IFN-g KO mice showed that Th1 cells control both themselves and the corresponding inflammation.Conclusion: Our results show that IFN-g is an important player in the regulation of a Th1 dependent inflammation. The proinflammatory cytokine can act as a negative feedback regulator and control the self-limitation of a Th1 dependent inflammation. Objective of the study: CD4 + regulatory T cells (Tregs) are increasingly recognised to play an important role in the maintenance of T cell homeostasis and self-tolerance. We recently reported a phenotypic and functional dichotomy among naturally occurring murine Treg subsets characterised by the expression of the integrin a E and CD25. a E -expression on Tregs correlates with an effector/memory-like phenotype, in contrast to naive-like a E -CD25 + Tregs. These phenotypic characteristics have a crucial impact on the migratory properties of Tregs in vivo and ultimately result in a differential potency to suppress immune responses within distinct anatomical compartments. We want to clarify whether a E + Tregs originate from the thymus as a distinct lineage or whether they are generated in the periphery under certain conditions. Materials and methods: We performed BrdU-labelling experiments to follow in vivo proliferation of different Treg subsets under normal and athymic conditions. In transfer experiments we investigated the stability of their phenotype and conditions of their generation.Results: The effector/memory-like phenotype of a E -expressing Tregs suggests antigen-specific differentiation or expansion in the periphery. Indeed, BrdU-labelling experiments in both normal and thymectomised mice revealed a high frequency of proliferating cells within this subset. Interestingly, a E -expressing Tregs were comparatively enriched in adult-thymectomised mice, as they sustained stable cell numbers even in the absence of thymic output, while mild lymphopenia was observed in the remaining T cell compartment.We transferred distinct Treg subsets into non-lymphopenic recipients and observed that under such conditions a E -expressing Tregs represent a stable subset or differentiation stage of Tregs rather than a transient population. By transferring TCR transgenic cells and subsequently applying antigen, we want to identify the cellular precursor of a E -expressing Tregs and the conditions of their generation in vivo. Initial experiments indicate that a E -expression is indeed induced on a fraction of transferred cells under tolerogenic conditions. Interestingly, this induction appears to be confined to certain compartments.Conclusion: a E -expressing effector/memory-like Tregs represent a stable population in the normal immune system. They contain cells, which are constantly cycling, presumably in response to self-antigen and/or environmental antigen. These effector/ memory-like Tregs have the potential to maintain a stable population size in the periphery even in the absence of thymic output. Preliminary results suggest, that this phenotype is induced in the periphery after antigen-specific differentiation under distinct conditions. A better understanding of the fundamental physiology of the generation of Treg subsets may aid in the design of future therapeutic strategies for the treatment of ongoing autoimmune diseases. Correlation between Histamine and Mast Cells and Presence of IgE. 1 1 Immunology, Shaheed Beheshti University, Medical School, Tehran, Islamic Republic of Iran.Aim: According to presence of immune system factors in chronic periapical lesions, hypersensitivity reactions may have some role in pathogenesis of these lesions. Up to now, among the factors participating in type I hypersensitivity reaction, only one of them has been studied. So the aim of this study was to evaluate the relationship between presence of IgE & number of mast cell and presence of IgE and histamine in human dental chronic periapical lesions.Materials and Methods: 40 specimens were collected from 39 patients. After extraction of the lesions, they were divided into two sections. Half of the sections were used for explant culture and were maintained for 3 days. Then sandwich enzyme linked immunosorbent assay (ELISA) was used to determine the presence and concentration of IgE and histamine. Based on the presence of IgE, the samples were divided into case (with IgE) and control (without IgE) groups.The other sections were used for estimating the number of mast cells by histopathological technique.Results: The average percent of mast cells in case and control groups was respectively, 10.25 F 7.02 and 6.9 F 4.09. Statistical analysis (Mann Whitney-U test and Spearman correlation coefficient) showed that there is no difference between the case and control groups regarding the number of mast cells and histamine concentration. Also there was not any correlation between IgE and mast cells or IgE and histamine.Conclusion: It is concluded that hypersensitivity reaction type I possibly does not have any role in pathogenesis of chronic periapical lesions. Neutrophil Chemotaxis and Dental Caries. 1 1 Immunology, Shaheed Beheshti University Medical Science, Tehran, Islamic Republic of Iran.Aim: In addition to microorganisms, several other factors such as host immune responses are also involved in pathogenesis of dental caries. Among the factors of immune system, the role of neutrophil in prevention or pathogenesis of dental caries is not well studied. So the aim of this study was to investigate the neutrophil chemotaxis in patients with dental caries.Materials and Methods: For this purpose, fifty of dental students with dental caries were selected. 10 ml of heparinized blood were collected from subjects for neutrophil chemotaxis test by modified Boyden chamber method.Results: The amount of neutrophil chemotaxis in the above subjects was 94.38 F 12.08 micrometer. Statistical analysis did not show any significant difference regarding to neutrophil chemotaxis in defferent degrees of DMF. But by comparing of neutrophil chemotaxis with the degree of active caries, significant differences were shown between 0 and 5 degrees; and 1 and 5 degrees of active caries regarding to the mean of neutrophil chemotaxis.Conclusion: It is suggested that not only deficiency in neutrophil chemotaxis is not assumed in pathogenesis of dental caries, but also there is a direct correlation between neutrophil chemotaxis and the degree of active caries. Of course more studies are needed in order to prove this hypothesis. Correlation between Neuropeptides Concentration and Different Parts of Human Gingiva and the Effects of Neuropeptides on Neutrophil Death. 1 1 Immunology Dept, Shaeed Beheshti University of Medical Science, Tehren, Islamic Republic of Iran.Objective: It is not well defined up to now that why some parts of gingiva is more susceptible to periodontal diseases. Since these regions are adjacent to the foramen of some nerves and paths and regarding to the important role of neuropeptides in inflammation and specially gingival and periodontal inflammation. On the other hand it was shown that substance P (SP) and calcitonine gene related peptide (CGRP) have some role in periodontal diseases. Since it was established that neutrophil is one of the most important defense line against periodontal diseases. So the aim of this study was to determine the correlation between concentration of neuropeptides and different region of human healthy gingival and their effects on neutrophil death.Materials & Method: In the first section twenty gingival samples from first maxillary incisor and molar regions and twenty gingival samples from other regions were collected during periodontal surgery. Tissue samples were immediately transported to sterile tubes filled with tissue culture media. After culturing for 72 hrs the supernatant fluid were extracted and after diving them in microtubes, were frozen at À70 degree of centigrade. EIA was used for detection of neuropeptides and in the second section we collected heparinate blood from a healthy individual neutrophil were collected from it. For this purpose, 5ml heparinate blood mixed with five ml dextran was maintained at laboratory temperature for 45 minutes and the high concentration and low concentration extract of neuropeptides (regarding to the concentration of SP and CGRP in first section) were collected with neutrophils Annexin V flous stanning kit was used for detecting the death of neutrophils under flourecence microscope.Finding: we find significant difference between different gingival regions regarding to CGRP concentration and it was also a significant reverse correlation between CGRP and different of Gingiva We could not find any significant correlation between SP and different regions of gingiva. Both SP and CGRP were present in the all of the samples. In the second part we find that SP and CGRP significantly induce apoptosis in neutrophils.Conclusion: It is concluded these neuropeptide probably participate in the regulation and maintance of gingival health and based on the low level of CGRP in the gingival of upper and lower 6 dental region prevalence of local aggressive priodontitis distributed to low level CGRP and SP in this region we could suggested a possible role for the susceptibility of these regions to localized priodontitis and in the second part of this study we can concluded that both of SP and CGRP induces apoptosis instead of necrosis in the neutrophil, this is also may be parallel with regulatory role of these neuropeptides. MyD88, the first adaptor protein that was discovered, possess a TIR-domain that interacts with a Toll-like receptor and with IL-1R, leading to activation of the NF-kB and JNKsignaling systems. MyD88 is highly conserved in nature. The murine MyD88 splicing has been established as the generator of MyD88s, there is not information yet on such human MyD88s processing. By nucleotide sequencing analysis of U937 and human peripheral blood mononuclear cells we noticed that human MyD88 splicing is comparable to that reported in the mouse. It therefore lacks the ID domain (aa 110-154). In the human mononuclear cells tested, MyD88s was constitutively expressed as a transcript and as a protein. However, the regulatory mechanisms in which they are involved could be exquisitely different. Background: Pediatric systemic lupus erythematosus (SLE) is a multisystem disease that significantly impacts quality of life (QOL) of children. SLE's impact on school attendance, an important outcome and potential predictor of other outcomes, has received little attention.Objective: Examine the relationship of school attendance with QOL, physical function, SLE activity and damage in children with SLE.Design/Methods: In this cross-sectional study, children with SLE (6-18 years) and parents completed child/parent versions of: Childhood Health Assessment Questionnaire (CHAQ), Pediatric QOL Inventory (PedsQL Generic 4.0 and Rheumatology 3.0 modules). Physician completed: SLE Disease Activity Index (SLEDAI) and Systemic Lupus International Collaborating Clinics/ACR Damage Index (SLICC). Number of days over the prior 30 the child missed school due to physical/mental health reasons was recorded using PedsQL family information form. Spearman correlations were determined between number of missed school days and other variables.Results: 24 children (23 girls) with SLE (mean age 15 F 3 years, mean education 9 th grade, mean SLE duration 46 F 30 months, SLEDAI 0-24, SLICC 0-6, median CHAQ 0.3, mean PedsQL-Generic 67 F 20), and 19 parents (median CHAQ 0, mean PedsQL-Generic 69 F 18) participated. 4 children were excluded because school attendance was inapplicable. Mean number of days too ill to play = 3 F 7 and mean number of days needed someone = 3 F 6 days. The number of missed school days moderately correlated with decreased QOL as reported by parents and as measured by the PedsQL-Generic module (r 0.56, p 0.02), but did not correlate significantly with Rheumatology module, CHAQ, SLEDAI, SLICC, or any of the child reported scores.Conclusions: Number of missed school days is correlated with decreased general QOL in children with SLE as perceived by their parents. However, parallel correlation between missed school days and overall QOL as perceived by children was not identified. Discrepant perception between parents and children warrant further investigation in a larger cohort. Lack of correlation of school attendance with other scales suggests that generic scale captures a less tangible element of SLE.F2.57. Improvement of Antimycobacterial Therapy Due to IL-10 Activity Blockage Is Strain Dependent.S. Roque, 1,2 C. Nóbrega, R. Appelberg, 1 M. Correia-Neves. 1,3 1 Laboratory of Microbiology and Immunology of Infection, Institute for Molecular and Cell Biology (IBMC), Porto; 2 Mestrado de Imunologia Clinica, Universidade da Beira Interior; 3 Instituto Superior de Saude do Alto Ave (ISAVE), Fontarcada, Portugal.Mycobacterium avium infection is a common opportunistic infection in immunocompromised patients. Antimycobacterial treatment implies a combination of two or more drugs administered for long periods. Furthermore, this chemotherapy repeatedly fails to induce sterile cure, and the occurrence of antibiotic-resistant bacteria is not a rare event. IL-10 is a cytokine with pleiotropic activities. IL-10 major effects are associated with its anti-inflammatory and immunosuppressive properties. Particular interest on IL-10 results from its ability to increase susceptibility to infection in several mouse models. In fact, previous studies from our group have shown that abrogation of IL-10 activity improves the efficacy of antimycobacterial drugs in BALB/c mice.Surprisingly, in the present study we show that this IL-10 effect seems to be strain specific. Combination of anti-IL-10 receptor mAb administration and antimycobacterial therapy (clarithromycin, rifampicin and ethambutol) in BALB/c and C57BL/6 chronically infected with M. avium (strain 2447) only increased response to treatment in the BALB/c strain. Whether this represents strain differences in the immune response to M. avium during antimycobacterial treatment is currently being investigating. Quality Control of DNA with the Agilent 2100 Bioanalyzer for Oligonucleotide Array CGH (aCGH). Samar Lightfoot, 1 Hans Brunnert, 2 Carsten Buhlmann, 2 Paige Anderson. 1 1 Agilent Technologies, Palo Alto, USA; 2 Agilent Technologies, Waldbronn, Germany.Comparative genomic hybridization (CGH) measures copy number variations at multiple loci simultaneously, providing an important tool for studying cancer and developmental disorders, and for developing diagnostic and therapeutic targets. We have recently developed an oligonucleotide array platform for array based comparative genomic hybridization (aCGH) analyses that can detect and map copy number alterations in the human genome, including single copy losses and gene specific homozygous deletions, as well as amplicons of varying sizes.The application of this technology to the study of human disease requires adequate quality control of DNA sample preparation prior to hybridization. The Agilent 2100 bioanalyzer and associated RNA assays are now established industry standards for checking the integrity of RNA samples. However, in addition to RNA sample analyses, the bioanalyzer has on-chip electrophoresis capabilities for DNA analysis. Here we investigated the use of the bioanalyzer for monitoring critical steps in the workflow of an aCGH experiment including DNA amplification, digestion of template and labeling. Our results demonstrates that the bioanalyzer can robustly monitor the quality and quantity of DNA templates used in aCGH experiments. Multiple sclerosis is a chronic autoimmune disease mediated by T cells reactive to different antigenic peptides of the myelin sheath. Experimental autoimmune encephalomyelitis induced with peptides such as MBP, MOG, PLP emulsified in CFA recapitulates the human disease in mice. T cells re-directed against the antigen-specific T-lymphocytes that mediate immunopathology have been previously shown to be selective and effective as a targeted therapy of experimental autoimmune encephalomyelitis (Mekala et al. To develop humanized receptors capable of re-directing therapeutic T lymphocytes against potentially disease-causing cells, we linked the immunodominant epitope, 84-102, of the human myelin basic protein MBP to the extracellular and transmembrane domains of the HLA-DR2 beta chain (DRB1*1501) and the TCR-zeta cytoplasmic domain. We similarly linked the DR2 alpha chain (DRA*0101) to the TCR-zeta. The alpha and beta chain constructs (denoted as HC2) were linked using the TMV 2A sequence and subcloned into the MSCV-I-GFP retroviral vector. GFP positive, retrovirally transduced 4G4 hybridomas and C57Bl/6J CD8 + T cells cells were isolated by flow cytometry. Western and flow cytometric analysis showed the expression of the construct. In order to assay the specificity of effector HC2 cells we used two different types of target cells: Ob hybridoma specific for human peptide MBP 84À102 /MHC II DR2 (courtesy of Dr. Lars Fugger, Skejby Hospital, 2rhus) and hMBP 84À102specific T cell line derived from humanized DR2xTCR doubletransgenic mice. Recognition of the chimeric receptor by hMBPspecific cells was shown by the production of specific cytokines (interleukin-2 and interferon-gamma) by the HC2-transduced T cells. Recognition of target cells by HC2-transduced T cells also induced proliferation of the transduced therapeutic cells. We have also shown that cytotoxic HC2 cells could specifically recognize the cognate TCR of MBP 84À102 -reactive target cells and kill the target cells in vitro. In vivo studies in a humanized murine model system, validating the use of these modified T cells as a cellular immunotherapeutic agent capable of selecting targeting pathologic antigen-specific T cells, are ongoing. LIF is a neurotrophic cytokine, belonging to the IL-6 family of cytokines. It plays a role in oligodendrocyte survival, but also in stem cell biology. Here we searched for possible immune functions of LIF.RT-PCR analysis proved the presence of LIF receptor beta on murine macrophages, dendritic cells and T-cells and its upregulation by activation. LIF -/-mice suffered from a less severe course of EAE in comparison to wild-type mice until day 70 p.i. In primary lymph node proliferation assays, lymphocytes from LIF -/-mice responded less to MOG 35-55 than those from wild-type mice, but exhibited a similar mitogen response. The defect in antigen-specific proliferation could not be restored by addition of LIF or IL-6 to cell culture. Supernatants from primary lymph node cultures were used to investigate cytokine production after immunization with MOG 35-55. LIF -/mice displayed decreased levels of MCP-1 and IFN-g whereas no difference in the production of IL-12, IL-6 or IL-5 was observed. Finally, a histological analysis was performed to investigate the inflammatory reaction in situ after immunization with MOG 35-55. The blinded quantification of CD3 and Mac-3 positive cells revealed significantly lower numbers of T-cells and macrophages in the spinal cord of LIF -/-mice on day 60 p.i. These results were paralleled by a decrease of MCP-1 and GM-CSF mRNA levels in the spinal cord of LIF -/-mice on day 40 p.i.In summary, our results are in line with a defective antigen-specific T-cell priming and a selective defect in cytokine/chemokine production in LIF -/-mice. This may result from impaired recruitment of antigen presenting cells (APC) and disturbed APC-T-cell interaction overall leading to a less severe course of MOG 35-55 EAE. The direct effects of LIF on T-cells are currently under investigation. Novel Immunotoxin: A Fusion Protein Consisting of Gelonin and an Acetylcholine Receptor Fragment as a Potential Immunotherapeutic Agent for the Treatment of Myasthenia gravis.M. Hossann, 1 Z. Li, 2 Y. Shi, 2 U. Kreilinger, 1 J. Buettner, 1 P. D. Vogel, 1 Y. Jingming, 2 J. G. Wise, 1 W. E. Trommer. 1 1 Chemistry, TU Kaiserslautern, Kaiserslautern, Germany; 2 Biotechnology, Shanxi University, Taiyuan, China.In continuation of our attempts for antigen-specific suppression of the immune system (Urbatsch, I.L., Sterz, R.K.M., Peper, K., and Trommer, W.E. 23, 774-779) a fusion protein composed of amino acids 4-181 of the extracellular domain of the a-subunit of the human muscle acetylcholine receptor and the plant toxin gelonin was expressed in E.coli.The fusion protein formed inclusion bodies but could be solubilized in the presence of guanidinium chloride. It exhibited a rather native structure as shown by antibodies recognizing a conformational epitope. Half maximal inhibition of translation was achieved at 46 ng/mL as compared to 4.6 ng/mL for native and 2.4 for recombinant gelonin.Its use as therapeutic agent for the treatment of Myasthenia gravis was investigated in an animal model. Female Lewis rats were immunized with complete acetylcholine receptor from the electric ray Torpedo californica and developed thereafter experimental autommune Myasthenia gravis. Quantitative assessment of the disease was achieved by repetitive stimulation of the sciatic nerve. Rats showed no more symptoms of Myasthenia gravis, neither visually nor electrophysiologically after treatment with the fusion protein as determined one and seven weeks after the second application. Fumarate Therapy Ameloriates Chronic Experimental Autoimmune Encephalomyelitis (EAE). S. Schilling, 1 S. Goelz, 2 R. A. Linker, 1 F. Luhder, 1 R. Gold. 1 1 Institute for Multiple Sclerosis Research, University of Goettingen and Gemeinnuetzige Hertie-Stiftung, Goettingen, Germany; 2 Biogen Idec, Cambridge, USA.Background: Treatment with fumaric acid derivates is well established as an effective therapy in severe psoriasis, a Th1 mediated skin disease. It has been proposed that one of the underlying ways that fumaric acid works is by inducing a Th1 to Th2 shift. In this study we investigated the clinical and molecular effects of Dimethyl fumarate (DMF) and Monomethyl fumarate (MMF) in chronic EAE, a model for multiple sclerosis.Methods: C57BL/6 mice were immunized with myelin-oligodendrocyte glycoprotein peptide aa 35-55 (MOG 33-35)/ CFA and received pertussis toxin. 3 groups of 8 mice/group were treated from day 3 to day 30 twice a day with 5 mg/kg body weight Dimethyl fumarate (DMF), Monomethyl fumarate (MMF) or the vehicle alone. Medication was administered by oral gavage. Animals were weighed and scored for clinical signs of disease on a daily basis over 30 days. Mice reaching paraplegia had to be sacrificed due to animal experimentation laws. Blood samples were taken from all mice before immunization, at the peak of the disease (day 11) and in partial remission (day 21), plasma protein concentration of 60 cytokines, chemokines and other markers was measured by Multi-Analyte Profile (MAP) testing.Results: In the control group and MMF treated group, 5/8 mice suffered from paraplegia, whereas in the DMF group only 2/8 mice reached this clinical endpoint. Onset of disease was earlier in the MMF treated group (mean: day 11,8) compared to DMF (mean: day 13,6) and control group (mean: day 12,3), this was not significant. DMF treated mice had a significantly less severe clinical course in this preventive treatment approach, pb0,001. Blood samples are currently analyzed using a Multi-Analyte Profile including 60 cytokines and chemokines, and histological studies are ongoing.Conclusion: Our data suggest that DMF has a beneficial effect in chronic EAE. We will present cytokine and histological results pending further analyses.Sa1.05. Beneficial Autoimmunity Restrains Destructive Immunity in a Regulatory Manner.Nathan Karin, 1 Yaniv Zohar, 1 Uri Weinberg, 1 Rachel Anunu, 1 Gizi Wildbaum. 1 1 Immunology, Rappaport Faculty of Medicine, Technion, Haifa, Israel.In previous studies we have shown that targeted DNA vaccines encoding selected proinflammatory mediators, particularly chemokines and cytokines, could effectively suppress experimentally induced autoimmune diseases in a selective manner. For example: targeted DNA vaccine encoding the CC chemokine RANTES (CCL5) could selectively suppress experimentally induced rheumatoid arthritis, but had no beneficial effect on experimental autoimmune encephalomyelitis (EAE), whereas DNA vaccines encoding MCP-1 (CCL2) effectively suppressed both diseases. The beneficial effect of each vaccine could be transferred by chemokine-specific autoantibodies developed in protected donors.Throughout these studies we have repeatedly observed an unexpected phenomena. The elicitation of beneficial autoantibody production, during ongoing autoimmunity, is very rapid. In the current presentation we shall show that this rapid response results from an amplification of a regulatory response induced by the disease itself. We shall show the high relevance of this regulatory mechanism to human disease and its therapeutic implications for autoimmunity and cancer.Key reference: Wildbaum, G., Nahir, M. & Karin, N. Beneficial autoimmunity to proinflammatory mediators restrains the consequences of selfdestructive immunity. Immunity, 19, 679-688 (2003 CD4 + CD25 + FoxP3 + regulatory T-cells suppress human and murine T-cell function. The role of regulatory T cells in human disease and its modulation are of major interest. Enhanced survival and expansion of regulatory T cells would be beneficial in autoimmune disease. In contrast, increased depletion by apoptosis would be advantageous in cancer. In addition to their described sensitivity to IL-2 deprivation, we show that freshly isolated FACSsorted human regulatory T cells are highly sensitive towards CD95dependent apoptosis in contrast to their CD4 + CD25-T cell counterparts. However, restimulation of expanded regulatory T cells revealed a reduced sensitivity towards activation induced cell death (AICD) in contrast to AICD-sensitive CD4 + CD25-T cells. Simultaneously, expanded regulatory T cells remained highly sensitive towards CD95L-triggered apoptosis. Murine CD4 + CD25 + regulatory T cells displayed a similar sensitivity. Our data suggest a model in which CD4 + CD25-effector T cells could modulate the number of regulatory T cells via the CD95L/ CD95 system in the contraction phase of an immune response. Furthermore, we found a defective suppressive function of regulatory T cells in Multiple Sclerosis (MS) patients. Given known alterations of the CD95 system in MS we are investigating whether an altered sensitivity of regulatory T cells towards CD95-dependent apoptosis could be critical for the modulation of defective regulatory response in Multiple Sclerosis.Sa1.07. Accumulation of CD4+CD25+ Regulatory T Cells in the CNS during Recovery from EAE. Many studies focus on the role of CD4 + CD25 + regulatory T cells (Tregs) in preventing the onset of autoimmunity. Here we have investigated the regulation of activated pathogenic T cells in a spontaneously remitting model of autoimmune disease-experimental autoimmune encephalomyelitis (EAE). EAE was induced by immunization of C57BL/6 mice with the central nervous system (CNS) autoantigenic peptide MOG(35-55). In our hands this leads to the development of EAE from 7 days post-immunization, with peak of disease in the second week, followed by a recovery phase with most mice free of clinical signs by 30 days post-immunization. Spinal cord and draining lymph node (LN) cells were purified from mice at different timepoints during disease. Cells were phenotypically examined by FACS analysis, and functionally tested for their ability to proliferate or suppress proliferation and cytokine production of naRve or primed cells in vitro. We found that the proportion of CD4 + cells expressing CD25 in the CNS increased during the course of disease, correlating with recovery. These CD4 + CD25 + cells preferentially produced IL-10, while IFNg-secreting CD4 + cells were CD25-. In vitro, CD4 + CD25 + CNS cells were anergic, but proliferated in response to anti-CD3 in the presence of IL-2. These CD25 + cells could also suppress proliferation of responder CD25-cells in vitro. Anti-CD25 (PC61) antibody was used to deplete CD25 + cells in vivo 3 days prior to EAE induction. This depletion led to exacerbated disease with greatly delayed recovery, supporting a functional role for Tregs in remission. Interestingly, CD25-depletion after recovery rendered mice fully susceptible to reinduction. Moreover, transfer of CNSderived CD25 + cells led to accelerated recovery from subsequent EAE. In conclusion, accumulation of CD4 + CD25 + Tregs (producing IL-10) in the CNS during the recovery phase of EAE suggests a role in the control of myelin-reactive pathogenic T cells. Our hypothesis is that autoantigen-reactive regulatory T cells are expanded or induced in draining lymph nodes, then recruited to the effector site to aid in the resolution of this autoimmune disease. Multiple sclerosis is defined as inflammatory demyelinating disease of the white matter of the central nervous system. While the functional neurological deficit of patients with acute or relapsing-remitting MS can be explained by the focal white matter lesions in the CNS, this is not the case for patients with primary or secondary progressive MS who experience a gradual accumulation of their clinical deficit. So far no pathological feature of the disease has been described which clearly distinguishes relapsing-remitting from progressive disease in MS patients.In the present study, we systematically analyzed cortical and white matter pathology in a large sample of multiple sclerosis brains with different disease courses (11 cases with acute MS, 6 with RRMS, 20 with SPMS and 14 with PPMS). Hemispheric and double hemisperic tissue sections were examined for cortical demyelination and pathological changes in the white matter which offered the unique opportunity to evaluate disease involvement of large tissue areas.Cortical demyelinated plaques were abundant in patients with primary or secondary progressive MS, but were virtually absent in patients with acute or RRMS (percentage of demyelinated cortical area: acute MS: 0,36; RRMS: 4,54; PPMS: 14,89; SPMS: 21,22; P = 0,0014). Focal load of demyelinated lesions in the white matter was almost the same in the four groups.Similar results were obtained from our analysis of the cerebellar cortex (percentage of demyelinated cortical area: acute MS: 0,85; RRMS: 1,89; PPMS: 33,28; SPMS: 38,2; P = 0,042).Especially in primary, but also in secondary progressive MS cases, myelin pallor was observed in the normal appearing white matter, which was associated with significant inflammation as well as microglia and macrophage activation. These pathological changes were sparse in acute and RRMS brains (inflammatory infiltrates per mm 2 : acMS: 0,05; RRMS: 0,07; PPMS: 0,20; SPMS: 0,30; P = 0,019). No correlation between size and location of white matter plaques and cortical demyelination or diffuse white matter damage was observed.In conclusion, we suggest that in MS brains three different pathological processes occur, which are are stage-specific and occur at least in part independently from each other: focal white matter plaques, cortical demyelination and diffuse damage of the white matter.All pathologies occur on the background of inflammation although the type of inflammation is different. Focal white matter lesions seem to be due to new waves of inflammation entering the brain with BBB damage. With chronicity of the disease, inflammatory cells accumulate gradually throughout the whole CNS, leading to diffuse damage in the bnormalQ white matter and the cortex. Using mouse models of distinct hereditary demyelinating neuropathies (heterocygous P0-deficiency, P0+-and homocygous CX32-deficiency, CX32-) we could recently demonstrate that T-cells and macrophages are involved in the pathogenesis of hereditary demyelinating disorders. Beyond these immune cells we have also found blood-derived CD34+ fibroblast-like cells in the endoneurium of P0+-mice. We supposed that these fibroblast-like cells might be identical to the recently described population of CD34+ peripheral blood fibrocytes and might therefore comprise another cell type of potential importance for immune regulation in hereditary demyelinating neuropathies.In this study we further characterized this novel cell population in the endoneurium of P0+-mice. We observed that these fibroblast-like cells show close contacts with endoneurial macrophages. Contact sites between these two cell populations were found regularly under normal and demyelinating conditions. The interaction between these two cell populations seems to play a role for immune regulation within the peripheral nerves since immunodeficient P0+-/RAGmice lack an age-related increase of CD34+ cells that parallels the occurrence of pathological changes within the peripheral nerves of P0+-mice. We suppose that this lack of CD34+ fibroblast-like cells might result in altered macrophage activation.In conclusion we could show that macrophages and fibroblastlike cells have close contacts within the endoneurium. According to our morphological observations we hypothesize that CD34+ fibroblast-like cells are involved in regulation of macrophage function under demyelinating conditions. Introduction: Increasing evidence indicates that infection with the Epstein-Barr virus (EBV) has a role in the pathogenesis of many human chronic autoimmune diseases, including multiple sclerosis (MS). We have proposed a new hypothesis that chronic autoimmune diseases occur in individuals genetically susceptible to the effects of B-cell infection by EBV, and that the EBV-infected B cells not only produce autoantibodies but also inhibit activationinduced apoptosis of autoreactive T cells in the target organ (Trends Immunol 24 (2003) 584-588; http://eprint.uq.edu.au/ archive/00001146). Decreased T-Cell Immunity to Epstein-Barr Aims: This study aims to determine whether patients with MS have an increased frequency of EBV-infected immortalized B cells and defective immunity against latent EBV infection, which may lead to the development of MS.Methods: Sera from patients with MS (not on immunomodulatory therapy) and healthy controls are tested for the presence of anti-EBV viral capsid antigen (VCA) IgG or anti-EBV nuclear antigen (EBNA) IgG using ELISA. We are using real-time PCR to quantify the EBV DNA load in the cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMC) of patients with MS. We are also performing IFNgamma ELISPOT assays to measure peripheral blood T-cell reactivity against major histocompatibility complex (MHC) class-I-restricted latent and lytic EBV peptides, MHC class-IIrestricted EBV peptides and human cytomegalovirus (HCMV) peptides.Results: Our preliminary results show detectable levels of EBV DNA in the CSF in 3 of 8 patients with MS. Furthermore, our results from the IFN-gamma ELISPOT assays indicate a reduction in the T-cell response to MHC class-I-restricted latent EBV peptides in MS patients compared to healthy controls.Conclusions: Our findings of the presence of EBV in the CSF of MS patients and of reduced T-cell immunity against latent EBV antigens may shed light on the role of EBV infection in the pathogenesis of MS.Sa1.11. CIDP is an inflammatory and demyelinating disease affecting the peripheral nervous system. It is related to the Guillain-Barré syndrome (GBS), but is a chronic condition and is distinguished from GBS by its temporal pattern and potential for clinical relapse. CIDP occurs more commonly in males than in females, and is thought to be autoimmune in nature, although this still remains unproven. Because it is thought that CIDP might have an autoimmune component, several studies have investigated HLA molecules carried by patients with CIDP. These studies have shown a trend towards increased carriage of HLA DR2 and DR3; however, since CIDP is relatively uncommon, the number of individuals studied has been small and the majority of patients have been male. Because studies in other autoimmune diseases have shown that sex-related factors appear to influence the risk associated with carriage of different HLA molecules, we set out to test a larger cohort of patients to determine if there are HLA associations with CIDP, and whether these differ depending on the gender of the patients.We have investigated carriage of class II HLA molecules in a cohort of 100 CIDP patients (60 male), and compared this to carriage of these molecules in 90 healthy controls and 71 patients with GBS. DNA was extracted from 5 ml whole blood, which was collected after written informed consent had been obtained. Dynal low-resolution SSP kits were used to type for HLA-DR, DQA and DQB molecules, to a resolution equivalent to that of serotyped subgroups.In comparison to female healthy controls, there was an increased carriage of DR2 by female patients with CIDP (P b 0.05), but not by female patients with GBS. Upon further subtyping of DR2 + individuals, the majority of individuals in all 3 groups were found to carry DRB1*1501. There were no differences in the frequency of carriage of DR2 between males in any of the 3 groups. There was a trend in male CIDP patients towards increased carriage of DR6 compared to male controls, whereas there was a trend towards decreased carriage of DR6 in female CIDP patients compared to controls. Our results did not confirm any association between carriage of DR3 and development of CIDP.There were no significant differences for frequency of carriage of DQA or DQB molecules between healthy controls, CIDP patients, or GBS patients when groups were considered as a whole, or subdivided based on gender.These results show that gender-specific HLA-DR associations occur in CIDP. They reflect similar findings in studies of other autoimmune diseases and provide additional evidence for an autoimmune disease mechanism in CIDP. The HLA typing in this study was only done to a low resolution, and further associations between CIDP (particularly in males) and carriage of particular HLA molecules may become apparent if the typing were done to the molecular level. Antibodies Specific for Myelin Proteolipid Protein Are of Potential Pathogenic Relevance in Myelin Opsonization in Multiple Sclerosis. J. M. Greer, 1 M. P. Pender. 1 1 School of Medicine, University of Queensland, Brisbane, Queensland, Australia.Myelin proteolipid protein (PLP) is the most abundant protein of central nervous system myelin and is a putative target antigen in multiple sclerosis (MS). Increased levels of T cell reactivity directed against PLP have been well-documented in MS. We have found that there are also increased levels of antibodies specific for PLP, particularly for the extracellular loop consisting of amino acids 180-230, in patients with MS compared to healthy controls and patients with other neurological diseases (OND). The aim of the current study was to determine whether these antibodies could have any pathogenic relevance in MS.Sera from 70 patients with MS (12 with primary progressive MS, 14 with secondary progressive MS and 44 with established relapsing-remitting MS or a first attack of MS), 25 patients with OND and 25 healthy controls were screened for their ability to opsonize DiI-labelled purified human myelin and thereby increase the uptake of the myelin into macrophages. The percentages of individuals in each group whose sera induced levels of myelin uptake greater than 2 standard deviations higher than the mean of the healthy control group were 69% for MS, 0% for healthy controls and 16% for OND patients. Over 70% of the sera from secondary or primary progressive MS patients induced increased levels of myelin uptake by macrophages. The percentage of patients with established relapsingremitting MS or a first attack whose sera induced increased levels of myelin uptake by macrophages was slightly lower (59%).Of those sera that induced increased myelin uptake, 60% contained antibodies specific for the PLP180-230 region, as assessed by ELISA. Preadsorption of sera with peptides from this region of the PLP molecule was found to be able to inhibit partly or wholely the opsonizing potential of those sera that showed PLP reactivity in ELISA. Thus, approximately 40% of patients with MS contain PLP-specific antibodies in their sera that can opsonize myelin for phagocytosis.Antibodies could have functional pathogenic effects in MS via a variety of mechanisms such as causing demyelination by antibody-dependent cell-mediated cytotoxicity or complementdependent lysis, or via opsonization of myelin resulting in phagocytosis. An antibody that could bind to extracellular epitopes of intact myelin (such as PLP180-230) would be more likely to be directly pathogenic than one that recognizes only disrupted myelin, although the later could be involved in escalating inflammation by activating the complement cascade. Our results show that PLPspecific antibodies can exhibit at least one of these potential pathogenic effects.Identification of MS patients exhibiting elevated pathogenic antibody responses may be an important step in selecting patients for treatment with intravenous immunoglobulin or plasmapheresis to decrease this activity. Autoantigen Specific T Cells Inhibit Glutamate Uptake in Astrocytes by Decreasing Expression of Astrocytic Glutamate Transporter GLAST-A Mechanism Mediated by Tumor Necrosis Factor-a. 1 1 Department of Neurology, Universitaet des Saarlandes, Homburg, Germany; 2 Stem Cell Section, Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA.Glutamate excitotoxicity is increasingly being recognized as pathogenic mechanism in autoimmune inflammatory disorders of the central nervous system (CNS). Astrocytes are the predominant players in clearing the extracellular space from glutamate and normally have extensive spare capacities in terms of glutamate uptake. We asked what might be the basis of glutamate accumulation in T cell-triggered autoimmune inflammation. In vitro, exposure of primary rat astrocytes to antigenactivated myelin basic protein (MBP)-specific T cells resulted in the decrease of astrocytic glutamate uptake rates as far as v max was concerned. In parallel, the amount of the astrocytic Na +dependent glutamate transporter GLAST was reduced in a time range of 48 to 60 h. Similar decreases of GLAST protein were observed in astrocytes re-isolated after co-culture with T cells activated by MBP and astrocytes as antigen presenting cells (APCs) or after co-culture with T cell blasts pre-activated in the presence of splenocytes beforehand. Since incubation of astrocytes with cell-free supernatant from MBP-activated T cells also resulted in a reduced expression of GLAST, a humoral factor appeared to be the driving agent. In blocking experiments using neutralizing antibodies and by exposure of astrocytes to recombinant cytokines, tumor necrosis factor-a (TNF-a) was identified to be responsible for the down-modulation of GLAST. GLAST was also down-regulated in the CNS of autoimmune encephalitic rats, but not in animals suffering from systemic inflammation. Since the loss of GLAST was not confined to inflammatory infiltrates, here too, a humoral factor seemed to be causative. In conclusion, T cell-derived TNF-a impaires glutamate clearance capacity of astrocytes in vitro and probably also in vivo in autoimmune inflammatory disorders providing a pathogenic link to glutamate excitotoxicity that may be responsible for early axonal dysfunction remote from active inflammatory demyelination. Full activation of T-cells requires costimulatory signals such as B7.1/B7.2-CD28/CTLA-4 interaction besides MHC-peptide-TCR signalling. We have previously shown in B7.1/B7.2deficient (DKO) mice that the genetic background (SJL/J versus C57BL/6J) determines the requirement for B7 costimulation in induction of EAE. Whereas, DKO on the B6 background were highly resistant to the development of EAE following immunization with the encephalitogenic MOG 35-55 peptide, DKO mice on the SJL mice remained susceptible to EAE when immunized with the PLP 139-151 peptide. Since the two strains differ in their MHC molecules, thus differ in peptide binding and the selection and expansion of the encephalitogenic T cell repertoire, we examined the role of the MHC as a determinant for the requirement of B7 costimulation inEAE. In order to detect further susceptibility regions we studied MOG 35-55induced EAE in 204 (SJLxB6) F2 DKO mice which were selected for at least one b allele at the MHC locus. No additional susceptibility regions were identified. Our results indicate that the expression of class II molecules (homozygosity of the s allele at the H2-A locus) is the major susceptibility factor which determines the requirement and dependence for B7-costimulation for the induction of EAE. Objectives: To induce tolerance in immune-competent animals with a plasmid (p4F10-eB7) expressing a membrane-bound single-chain antibody against CTLA4. Materials and Methods: Thirty 9-week old immune-competent C57B/L6 mice were divided into three groups. Ten mice received an intramuscular injection of endotoxin-free plasmid, pCBLacZ, at the left anterior tibialis. The plasmid encodes a potent immunogen, Escherichia coli beta-galactosidase. Another ten mice received five consecutive intramuscular injections of the plasmid. The other five received coinjection of two plasmids. Results: For single injection, we examined the expression of beta-galactosidase in muscle fibers on the 6 th and 12 th days after injection. Significant difference of transduced muscle fibers was only found 6 days after single injection. However, serum IgG against beta-galactosidase was much lower in the animals received consecutive injections of two plasmids as compared with those in the control group. Conclusions: Expression of anti-CTLA4 antibody in muscle cells protected the expression of beta-galactosidase in immune-competent mice. This membrane bound single-chain antibody attenuated host humoral immune responses during repeated immunization. The probable mechanism is peripheral tolerance induced by anergy of activated T cells after interaction with the single-chain antibody. This novel strategy targeting on activated T cells may be used for treatment of acquired autoimmune diseases.Sa1.16. Memory CD4+ T Cells and EAE. The memory T cell pool functions as a dynamic repository of antigen-experienced T lymphocytes that accumulate over the lifetime of the individual. Previous studies indicate that T cells which are reactive to myelin antigens are mainly memory cells in MS patients. We hypothesize that there are specific mechanisms of disease mediated by memory T cells. Here, we developed an animal model of memory CD4+ T cell-inducing EAE. Wild-type (WT) or MOG TCR transgenic memory CD4+ T cells were generated from WT B6 or 2D2 transgenic mice respectively, immunized with MOG/CFA for more than 100 days. CD4+CD44hi cells were sorted and immediately transferred into TCR ab -/-mice followed by immunization with MOG/CFA. A control group of mice immunized with MOG peptide were used to generate CD4+CD44hi effector cells. Our data indicate that disease induced by memory cells is more severe when compared to that induced by effector cells. Confocal double-labeling images showed CNS infiltration of CD4+ memory cells associated with astrocytic activation and dramatic demyelination as shown by GFAP-positive cells and SMI32 staining, respectively. Furthermore, sorting of memory CD4+CD44hi according to the expression of either CD62L or CCR7 providing central (TCM) or effector (TEM) memory T cells demonstrated more severe EAE induced by TEM than TCM associated with increase proliferation and high production of IFNg in response to MOG peptide in vitro. Costimulatory signal blockade of T cells in this model, demonstrated that while CTLA4Ig suppressed effector CD4+CD44hi-induced EAE, it did not significantly inhibit disease induced by memory CD4+CD44hi cells. By contrast, anti-ICOS-L treatment worsened effector cell-induced EAE, but inhibited memory cell-induced disease. Our data suggest the presence of specific mechanisms of disease mediated by memory CD4+ cells and has relevance to the treatment of human autoimmune disease by costimulatory signal blocking agents.Sa1.17. Therapeutic Effects of Glycosylated B Interferon (BIFN) on Childhood Adrenoleukodystrophy (ALD CCER). G. A. Moviglia, 1 A. E. Pereyra, 1 G. S. Shuster, 2 C. Ruggilo. 2 1 Immunotherapy, Regina Mater Foundation, Buenos Aires, Argentina; 2 MRI, Medical Image, Buenos Aires, Argentina.Childhood Cerebral Adrenoleukodystrophy (ALD CCER) is a genetic disease. Based on some bibliographic research and our own experience we conjecture that the lesions it causes in the central nervous system (CNS) are the result of an autoimmune demyelinating reaction resembling the Multiple Sclerosis (MS) inflammatory reaction. bIFN has shown a positive therapeutic action on MS; for that reason, we developed a clinical trial to test its effect on ALD CCER. Thirteen boys affected by the disease were divided into three groups: Group A, 5 patients with a Behavior Performance Index (BPI) of over 80 points, received bIFN; Group B, 5 patients with a BPI of under 80 points, received bIFN; and Group C, 3 patients with a BPI of under 80 points, did not receive medication. The bIFN was supplied by Ares-Serono International. The dosage schedule was 3 to 6.106 IU of bIFN by intramuscular injection, at 8 p.m. on three consecutive days every week. The patients were followed for 24 months via monthly neurological, psychological and immunological evaluation and by telephone follow-up thereafter to assess survival and general clinical and neuropsychological performance. The results of our small clinical trial strongly suggest that bIFN can change the natural history of ALD CCER. bIFN improved the clinical conditions of 4 out of 5 patients from the first group for almost three years, and has maintained these conditions for two of these patients up to the present (six years after stopping the bIFN treatment). MRI, immunological and psychometric data confirmed these findings, which support the theory that the interaction of immune cells with nervous tissue cells plays a crucial role in the CNS repair, as well as that bIFN may be a therapeutic agent for it. Multiple Sclerosis (MS) is the most common Neurological Autoimmune Disorder diagnosed in young adults and is characterized by the repeated occurrence of demyelinating lesions within the central nervous system (Pohlau, et al., 1998) .Part of the mechanism of MS is an abnormal production of auto-antibodies, specifically producing cross-reactive epitopes, which leads to an auto-immune reaction against myelin.It is our belief that IVIG can act as a neuro-immune modulator for patients diagnosed with MS. Current research has demonstrated that, in relapsing MS, an association exists between high dose IVIG and reductions in relapse rates, exacerbations and the number and size of brain lesions, as well as increases in functional improvements.A recent study from researchers at the University of California found that the anti-myelin antibody is found in virtually all patients with progressive multiple sclerosis (Brown, 2002) . We theorize that the anti-myelin antibody may be used as a marker for the treatment of patients diagnosed with progressive MS on high dose IVIG treatment.In our study, we report on a 43-year-old patient with a diagnosis of progressive multiple sclerosis; the disease that has resulted in rapid regression over the past 5 years. The subject presented to our clinic with a history of countless treatmentTs and therapies that failed to improve his condition or stall his regression. An extensive immune work-up revealed extremely low CD3 and CD8 numbers along with low lymphocyte stimulation function, an IgG3 subclass deficiency and positive anti-myelin antibodies; past records indicated the presence of positive anti-myelin antibodies for over 5 years. Upon completion of a thorough physical examination and work-up, the patient was started on high dose IVIG: 2g/kg divided over three consecutive days. Subsequent testing, conducted after a short period of time, revealed negative anti-myelin. As a result of this treatment, he is beginning to display increased energy, decreased muscle spasticity and improvement in his ability to walk.Based upon the preliminary data, we suggest that: 1) IVIG may have an immuno-modulatory effect in MS and 2) anti-myelin AB may serve as a marker for the immuno-modulation effect. Cross-reactive activation by high affinity non-self ligands, of T cells with the potential to recognize self, may be important in breaking self-tolerance. In a TCR transgenic mouse that shows a broad cross-reactivity to a number of ligands, we have previously shown that a hyperstimulating superagonist ligand, L144 induced unresponsiveness to a self-ligand, proteolipid protein (PLP) 139-151 peptide. In a further examination of the role of cross-reactive superagonists in an autoimmune response, we demonstrate here that a superagonist ligand can break tolerance induced by the lower affinity cognate antigen. In our proposed balteredQ hierarchical anergy model, T cells tolerant to cognate ligand, Q144, responded to superagonist, L144 by proliferation and the production of mainly IL-4 and IL-10 in vitro. In contrast, T cells that were tolerized to the superagonist, were unable to respond to any peptide that cross-reacted with the transgenic TCR. In vivo, low-dose immunization with superagonist was able to break tolerance to the cognate ligand, and resulted in a blunted proliferative response and Th2 cytokine production following a re-challenge in vitro. A combination of different T cell responses may thus provide a multilevel defense against autoimmunity during a T cell response against cross-reactive high-affinity foreign antigens, and may serve as an important regulatory mechanism to prevent autoimmune disease. T Cell Tolerance Sa1.20. Agonistic Anti-CTLA4 Antibody Inhibits T Cells Expansion, Cytokine Production and Development of Autoimmunity.K. 1 1 Center for Neurolosic Diseases, BWH, Harvard Medical School, Boston, MA, USA; 2 Pathology, BWH, Harvard Medical School, Boston, MA, USA; 3 Neurology, Kinki University, Osaka-Sayama, Osaka, Japan.We generated a novel agonistic anti-CTLA4 monoclonal antibody, by immunizing Lou/M rats with the CTLA4Ig fusion protein and screened the hybriboma supernatants for binding to CTLA4 in ELISA and CHO expressing CTLA4 on their surface. Of the three antibodies thus generated, one antibody 3C1.B5 was agonistic in that inhibited T cell expansion and IFNg secretion in vitro and in vivo. This contrasts with the previously described anti-CTLA4 antibody 4F10, which promotes T cell clonal expansion and IFNg secretion presumably by blocking the inhibitory signal delivered by CTLA4. We compared the effects of both anti-CTLA4 antibodies in murine relapsing remitting autoimmune disease experimental autoimmune encephalomyelitis (EAE), a demyelinating disease mediated by PLP139-151 specific CD4+ T cells in SJL /J mice. Whereas, administration of the 4F10 antibody in vivo exacerbated EAE, the agonsitic anti-CTLA4 antibody 3C1.B5 ameliorated disease. To understand the molecular basis for the difference in the functional effects of the two antibodies, we undertook epitope mapping and binding studies. We demonstate that the Tyrosine residue in the MYPPPY domain of the CTLA4 utilized for binding by the B7 molecules, is also crucial for binding of the blocking anti-CTLA4 (4F10) antibody but not for the agonistic anti-CTLA4 antibody (3C1.B5). Thus binding of the two anti-CTLA4 antibodies to a closely related but distinct epitopes gives distinct functional T cell responses. Tumor necrosis factor-a (TNF-a) as an important immune regulator and inflammatory cytokine is implicated in the pathogenesis of multiple sclerosis (MS). A single nucleotide polymorphism, G to A, at position À308 in the promoter has been previously shown. This polymorphism is associated with TNF-a production level. To study the effect of this polymorphism on susceptibility to multiple sclerosis, we screened genomic DNA samples from 98 definite MS patients and 97 unaffected ethnically matched individuals as healthy controls, using sequence-specific primers (PCR-SSP). Our findings suggest that polymorphism at position À308 and production levels of TNF-a could influence susceptibility to MS. Corticotropin-releasing hormone (CRH), a hypothalamic neuropeptide, is thought to have peripheral pro-inflammatory effects outside the central nervous system. Studies have shown that T lymphocyte can produce CRH, and CRH binding site presents on macrophages and lymphocytes. Upregulation of CRH peptide production has been shown in peripheral sites with acute or chronic inflammation. Our objective was to study the peripheral role of CRH on T lymphocyte and antigen-presenting cell (APC) activation in an immune mediated disease model of experimental autoimmune encephalomyelitis (EAE). Material and Method: Crh -/mice on a C57BL/6 Â 129 genetic background were immunized with MOG 35-55 peptide together with complete FreundTs adjuvant and pertussis toxin. The severity of EAE was scored on the scale of 0-5 as described previously. To eliminate the anti-inflammatory effect of corticosterone, astressin, a peripheral CRH antagonist with no glucocorticoid inhibition effect was given to wild-type mice on day 11-16 post immunization. In comparison between crh -/and crh +/+ T cells, T cell proliferation assay and the ELISA assay of cytokine production were conducted on either naRve CD4 + T cells with anti-CD3/anti-CD28 TCR stimulation or MOG-specific CD4 + T cells from immunized mice in response to g-irradiated MOG-loaded APC stimulation. The antigen presentation function was examined by MOG-specific wild-type T cell proliferation in response to MOG-loaded crh -/or crh +/+ APCs. Anti-CD3/anti-CD28 stimulated InBa phosphorylation in crh -/splenocytes was tested by western blot analysis. Result: We found that crh -/mice as well as astressin treated wild-type mice are resistant to EAE with decrease in clinical score as well as decreased cellular infiltration in the central nervous system. Furthermore, antigen-specific responses of primed T cells as well as anti-CD3/anti-CD28 TCR costimulation response of naRve T cells were decreased in crh -/mice with decreased production of Th1 cytokines, IFN-g and IL-2 and increased production of Th2 cytokine IL-5. Wild-type mice treated in vivo with a CRH antagonist astressin showed a decrease in IFN-g production by primed T cells in vitro. This effect of CRH is independent of its ability to increase corticosterone production, since adrenalectomized wild-type mice had similar disease course and severity as control mice. Furthermore, a decreased antigen-specific T cell response was observed in wild-type MOG-specific T cells when incubated with MOG-loaded crh -/-APCs compared with MOGloaded crh +/+ APCs. We found that InBa phosphorylation induced by TCR cross-linking was delayed and compromised in crh -/-T cells. Conclusions: Peripheral CRH exerts a pro-inflammatory effect in EAE with selective increase in Th1-type responses. This effect CRH is likely the result of optimal activation of T cells through activation of NF-nB pathway. Rationale: Oral HMG-CoA reductase inhibitors differ in their ability to treat hypercholesterolemia. Recent studies have shown that certain statins can promote a Th2 bias and prevent or reverse relapsing and chronic paralysis in experimental autoimmune encephalomyelitis (EAE). However, it is not clear whether some statins are more effective than others in treating this autoimmune disease. Furthermore, some studies have shown purified statin administered either orally or parenterally (i.p.) In this study we examined whether statins differ in their ability to modulate autoimmunity in EAE. We further investigated whether differences in formulation (prescription vs. purified) and route of administration (oral vs. parenteral) effect treatment and the development of Th2 bias. Prescription formulation of atorvastatin, rosuvastatin, lovastatin, simvastatin, pravastatin, and placebo was administered orally in PBS vehicle one time daily. Prescription form was administered at one and ten times the equivalent of the highest-approved, human dose. Purified atorvastatin was administered orally or parenterally. Th1 and Th2 cytokine profiles of T cells isolated from treated mice were evaluated by ELISA. Phosphorylation of STAT signaling molecules was measured by Western blot.Results: Control and placebo-treated mice developed EAE with similar incidence and severity. While all statins tested ameliorated EAE, atorvastatin and rosuvastatin were the most effective when administered orally in prescription formulation. All statins inhibited antigen-specific proliferation, secretion of Th1 cytokines and IL-12-dependent STAT4 phosphorylation. Atorvastatin and rosuvastatin induced Th2 cytokines, which was associated with increased phosphorylation of STAT6. Immunodulatory effects were reversed by treatment with L-mevalonate. Orally administered prescription atorvastatin was superior to purified atorvastatin administered either parenterally or orally.Conclusions: Our results show that all statins can ameliorate EAE progression, although individual statins appear to vary in the strength of their immunomodulatory effects and only some statins induce a Th2 bias. Atorvastatin and rosuvastatin, the most potent of the statins in lowering cholesterol, are the most robust in both amelioration of EAE as well as induction of a Th2 T cell phenotype, which likely reflects differences in enzymatic binding efficiency, half-life, and lipophobicity. Drug formulation and route of administration are important factors in determining in vivo immunomodulation by atorvastatin. This study highlights the importance of selecting the type of statin for testing in treatment of multiple sclerosis and other autoimmune diseases. Recent work using functional magnetic resonance imaging (fMRI) indicates that patients with multiple sclerosis (MS) show altered brain activation on motor and cognitive tasks relative to controls. However, few studies have examined MS patientsT brain activation patterns on fMRI probes of executive cognitive functioning. The present study used a modified version of the Tower of London task to examine potential alterations in the neural circuitry of strategic planning in MS. Six MS patients and four healthy controls were administered an fMRI Tower of London task, which included easy (2-3 move solution), hard (4-5 move solution), and control conditions. The hard greater than easy contrast was of particular interest in our analyses. fMRI data were obtained on a 1.5T GE scanner. Data were analyzed using a random effects model in SPM99. While both MS participants and controls activated predicted regions during task performance, including right dorsolateral prefrontal cortex, MS patients showed increased anterior right hemispheric activity relative to controls (P b .01, k=3). In contrast, controls tended to show more posterior right hemispheric activation than patients (P b .01, k=3). Behaviorally, controls tended to perform better than patients across task conditions. These preliminary findings support previous reports of altered brain activation patterns associated with cognition in MS. Our group and others are engaged in longitudinal research to determine the relationship between changes in neural activity and cognitive task performance, and to examine the potential utility of fMRI for tracking progression of brain changes associated with cognitive decline in MS. Sa1.25. A New Clinically Relevant Approach To Expand Myelin Specific T Cells. Nathalie Arbour, 1 Rejean Lapointe, 2 Philippe Saikali, 1 Jack P. Antel. 1 Intro: Human self-reactive T cells are postulated to play an important role in many autoimmune diseases. Although detection of self-reactive T cells directly ex-vivo has been achieved, in-depth functional characterization of these cells is impeded by their low precursor frequency. Hence, researchers have expanded self-reactive T cells in vitro in order to characterize and compare these cells from autoimmune patients and controls. Antigen specific T cells need to be re-stimulated on a regular basis with new antigen presenting cells (APCs) and antigen (Ag). This has been usually achieved by using autologous fresh or frozen irradiated peripheral blood mononuclear cells (PBMCs) or EBV-immortalized B cells or dendritic cells in the presence of Ag. This approach requires either one large blood sample from which cells are frozen for later use or repetitive blood draws. Evaluation of self-reactive T cells from many donors is thus hindered by the large volume of blood necessary. We explored a method successfully applied for tumor antigens to present myelin antigen to T cells using in vitro expanded autologous B cells (Lapointe et al. 2003) .Our goal is to assess the feasibility of expanding human myelin specific T cells from one small blood draw.Methods: Myelin specific T cells were expanded from 50-75 ml of blood. PBMCs were put in culture with myelin basic protein (MBP) or myelin oligodendrocyte glycoprotein (MOG) peptide. A week after the first round of stimulation T cells were separated into CD8 + and CD4 + using beads (Miltenyi) prior to be re-stimulated. In parallel, a fraction of donorTs PBMCs was stimulated with irradiated CD40L-expressing fibroblasts in the presence of IL-4; B cells proliferated and after few rounds of stimulation were the only cell type in the culture and expressed high levels of MHC molecules and co-stimulatory molecules, essential hallmarks of efficient APCs. Expanded B cells were loaded with Ag, irradiated and then used as APC to stimulate T cells for multiple subsequent rounds of stimulation. CD4 + T cell lines were re-stimulated every 10-14 days and CD8 + T cell lines every 7-9 days. Proliferation was measured by thymidine incorporation and cytokine release by ELISA.Results: At the second or third round of stimulation T cells were tested for their specificity by comparing their proliferation and IFN-g secretion in the supernatant when put in cultured with Ag-loaded B cells vs. unloaded B cells. T cell lines exhibiting Agspecific proliferation and/or IFNg secretion at least two times higher than in the absence of Ag were considered Ag-specific and were expanded further. MBP and MOG specific CD4 + and CD8 + T cell lines were obtained from multiple donors in comparable number than those obtained with a traditional approach (fresh PBMCs) and kept in culture for many weeks.Conclusion: Human myelin specific T cell lines both CD4 + and CD8 + can be expanded in vitro from a relatively small blood sample facilitating immunological studies of such cells in multiple donors. Objective:To investigate the effect of therapy with IL-15Fc and IL-2Fc fusion proteins on experimental autoimmune encephalomyelitis(EAE). Background: Peripheral T cell activation and their migration into the central nervous system(CNS) are involved in the pathogenesis of EAE.Targeting the IL-2 receptor, which shares the gand h portions with the IL-15R, suppresses immune responses.The IL-15Ra chain is specific for IL-15, and is expressed on CD8 + , natural killers, macrophages,endothelial cells and tissues such as brain, spleen and thymus. We used two fusion proteins to induce protection in EAE: IL-2Fc that binds to activated T cells and induces cytolysis, and IL-15Fc that binds to the IL-15Ra. Methods: C57BL/6 mice were immunized with MOGp35-55 in CFA to induce EAE. Daily i.p injections of either IL-2Fc (5ug/ dose), IL-15Fc (5ug/dose), IL-2Fc+IL-15Fc or control IgG2 were given for 10 days, starting either on day 0 or on day 10 postimmunization, to target the priming or effector stages of disease, respectively. Outcomes measured included clinical disease, cell proliferation measured by thymidine incorporation, cytokine profiles measured by ELISPOT assays, and immunohistological staining of the CNS at day 15, 30 and 40 post immunization. Results: Early treatment with the combination fusion proteins decreased disease severity(MMG 0.6; pb0.0001) and incidence(P = 0.01)significantly compared to the control Ig group (MMG=2.4). Treatment with any of the infusion proteins induced a high background proliferation of splenocytes; but MOG peptide-specific proliferation was suppressed in splenocytes from IL-15Fc(P = 0.002) as well as combination therapy (Pb0.0001) treated mice. Staining for inflammatory cells in the CNS showed a decrease in CD4 + and macrophage infiltrates in the protected groups, particularly in those receiving combination therapy. These scarce infiltrates were positive for IL-4 and IL-10. Protection is associated with an increase in Th2 cytokines both in the periphery and the CNS. The additive effect of combination therapy may be due to the cytolytic effect of IL-2Fc on MOG-specific T cells, and decreased cell migration into the CNS mediated by IL-15 blockade. Further studies need to be done in order to fully understand the mechanisms by which these molecules protect in EAE. Oral exposure of soluble antigens leads to the induction of systemic hyporesponses, a phenomenon known as oral tolerance. In this study, we isolated dentritic cells (DCs) from the PeyerTs Patches (PPs), mesenteric lymph nodes (MLNs) and spleens of fed mice and measured their ability to support CD4+ T cell proliferation, cytokine production and differentiation. We found that the ability of DCs to support T cell proliferation appeared in PPs first, followed by MLNs and spleen. Moreover, PP DCs promoted significant increases of Th1 cytokines (IL-2, IFNg and TNFa) but no increases of Th2 cytokines (IL-4 and IL-5). However, MLN DCs promoted significant increases of both Th1 and Th2 cytokines. Although DCs from all these lymphoid tissues were able to induce naRve T cells to express surface TGFb, as determined by the up-regulation of latency-associated peptide (LAP+), MLN DCs were the most efficient. At their peak time (~12hrs after last feeding), MLN DCs induced a~5-fold increase of CD4+LAP+ cells. These studies demonstrate that MLN DCs acquire fed antigens and induce the expansion of CD4+LAP+ T cells in situ which then may participate in specific tolerance to fed antigens and regulation of autoimmune processes associated with oral tolerance to autoantigens. Multiple Sclerosis (MS) is a chronic autoimmune disorder that affects the central nervous system. Although the aetiology of the progressive neurological loss has not yet been fully elucidated, it is believed that genetically determined susceptibility and environmental triggers are both implicated in the detrimental immune response against components of the myelin sheath, where myelinspecific T cells are thought to play a central role. As an animal model for this disease, Experimental Autoimmune Encephalomyelitis (EAE) can be induced in C57BL/6 mice. EAE is an inflammatory demyelinating disease that is primarily mediated by CD4 + T cells and shares most of the clinical and histopathological aspects of MS. Due that dendritic cells (DCs) are professional antigen presenting cells important for the activation of self-reactive T cells, we are interested in evaluating their therapeutic potential in the regulation of autoimmune responses. DCs have a central role in maintaining peripheral tolerance to self and alterations in their physiology are likely to be responsible for defective immune regulatory mechanisms. We first observed that immature DCs are able to promote tolerance in the EAE model. To further enhance the tolerogenic capacity of these cells, we used drugs that interfere with NFkB activity, such as andrographolide, a bicyclic diterpenoid lactone and Rosiglitazone, a PPARg agonist. In vitro, these molecules were able to interfere with DCs maturation and with their ability to present antigens to T cells. T cell activation by DCs was completely abolished by exposing DCs to andrographolide and Rosiglitazone during antigen pulse. Injections of immature DCs treated with either andrographolide or Rosiglitazone showed an enhanced capacity to reduce EAE symptoms. Our results indicate that injection of immature DCs can prevent myelinspecific T cells activation in EAE. These data suggest that pharmacological approaches that promote a tolerogenic phenotype in DCs could be useful as a potential strategy in the design of new therapies to prevent or treat detrimental immune responses. In SJL/J mice, PLP139-151 is the immunodominant encephalitogenic epitope and induces a relapsing-remitting form of EAE. It has been reported that CD4+CD25+ regulatory cells play a role in affecting the onset and progression of EAE, as transfer of CD4+CD25+ cells at these stages ameliorate disease.The role of CD4+CD25+ regulatory cells in the natural recovery from disease has not been well defined. Here we show that EAE-recovered SJL/J mice have an increase number of Forkhead box P3 (Foxp3)-expressing CD4+CD25+ T cells. These cells were anergic and inhibited the proliferative response of CD4+CD25-T cells against PLP139-151 or anti-CD3 stimulation. Depletion of CD4+CD25+ T cells prior to the recovery phase exacerbated disease, resulted in the expansion of IA s /PLP139-151-tetramer-positive cells and enhanced IFN-g production. In addition, transforming growth factor (TGF-h) was shown to be involved in the recovery from EAE as the percentage of CD4+CD25+ cells expressing TGF-h latency associated peptide (LAP) on the cell surface increased significantly in blood and spleen of EAE-recovered mice as compared to the naRve mice (P b 0.001 and P b 0.01, respectively) and in vivo neutralization of TGF-h abolished recovery from disease. Our results demonstrate that CD4+CD25+ regulatory cells mediate recovery from PLP139-151-induced EAE in SJL/J mice in which TGF-h plays an important role. Multiple Sclerosis (MS) is a multifactorial, polygenic disease that manifests itself as a chronic inflammation of the CNS. In addition to influencing the presence or absence of disease, genetics may also play a significant role in individual variations in prognosis and differential response to therapy among patients.In order to better understand the genetic variation involved in these heterogeneous aspects of the disease we selected 43 SNPs from 22 genes that were associated with risk or severity of MS in at least one published study. These loci were genotyped using DNA from a large MS patient registry which was established as a longitudinal study aimed at finding biomarkers of disease risk, prognosis and response to therapy.Our case-control association analysis utilized 190 patients enrolled to date and an ethnically-matched cohort of healthy controls (n = 363 When we compared patients with benign vs. malignant disease course (n = 37 vs. 27) we observed an association of MBP with disease prognosis (p=0.003, OR=5.319, CI 95 =1.727-16.393). Paradoxically, the DR15 allele also associated with the presence of benign disease (P = 0.004, OR=5.447, CI 95 =1.628-17.945); it is therefore a risk factor, while at the same time providing a better prognosis.A comparison of responders vs. non-responders to betainterferon or glatiramer acetate therapy (n = 38 vs. 28) showed a significant association of IL1B with response (Pb0.1, OR=5.182, CI 95 =1.821-14.747). Linear regression analysis of genotypic association with age of onset revealed a potential involvement of TNFSF6 (P = 0.002).All analyses were controlled for gender. Among several markers that exhibited significant sex-specific associations, TNF was associated with malignant disease only in female patients (P = 0.01), and TNFSF10 was significantly associated with risk of disease in males (P = 0.0016).These results illustrate the complex genetic etiology of MS, which involves a variety of sometimes gender-specific factors combining to influence not only the presence but the progression of the disease. These markers may eventually improve our understanding of the disease and provide better clinical and diagnostic indicators of disease risk and prognosis. Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). Although the cause of MS is still uncertain, an ongoing autoimmune response against myelin antigens seems most likely. Myelin Oligodendrocyte Glycoprotein (MOG) is considered the most promising candidate autoantigen in MS. MOG induces experimental autoimmune encephalomyelitis in several rat and mouse strains. However, studies in humans and animal models are based on a fragment of the MOG protein expressed in E. coli. To achieve physiological MOG expression with proper glycosylation, we cloned the human MOG isoform 2 gene in a lentiviral expression vector. We transduced primary cells and several human and murine tumor cell lines. Transduced cell lines expressed high levels of MOG on the surface. Stable expression was achieved in murine lymphoma and several human glioma cell lines. These transgenic lines allowed fast and reliable detection of anti-MOG antibodies at lower nanogram concentration comparable with the sensitivity of ELISA assays. Further studies are ongoing to determine anti-MOG antibodies in MS patients and controls to obtain further insights into the role of anti-MOG antibodies in multiple sclerosis. Role of CD46 in Multiple Sclerosis. A. L. Astier, 1 D. A. Hafler. 1 1 Center for Neurologic Diseases, Brigham and WomenTs Hospital, Boston, MA, USA.CD46 acts as a costimulatory molecule for human T cells and coligation with CD3 promotes T cell proliferation. Furthermore, addition of IL-2 induces a Tr1 phenotype, characterized by a large production of the regulatory cytokine IL-10. However, in a murine transgenic model, the two intracytoplasmic isoforms of CD46 (Cyt1 and Cyt2) that are produced by alternative splicing, have antagonist roles in an in vivo T cell-mediated inflammation. This difference has been linked to their differential effects on CD4+ T cell proliferation, CD8+ CTL cytotoxicity, as well as IL-2 and IL-10 production. Hence, CD46 is a crucial regulator of T cell activation, and depending on the ratio of the intracytoplasmic isoforms present, a different outcome in T cell activation might result in humans. As multiple sclerosis (MS) is an autoimmune disease with a direct involvement of T cells, we investigated the role of CD46 in MS. While as previously described, IL-10 could be induced upon CD46 stimulation in healthy donors (5/5), T cells from most patients with MS did not produce IL-10 or to a much lesser extend (8/11) . We then investigated the ratio of the two intracytoplasmic isoforms of CD46 in T cells of patients with MS as compared to healthy donors. Interestingly, we found that upon CD46 costimulation the Cyt1/Cyt2 ratio is altered in MS patients when compared to healthy individuals, which might correlate with the lack of IL-10 secretion observed upon CD46 stimulation. These preliminary data suggest that a dysregulation of CD46 activation in human T cells may be involved in MS etiology. Most autoimmune diseases disproportionately affect women. Unfortunately, most animal models and in vitro studies show that steroid hormones (estrogens and androgens) are suppressive for autoreactive responses. This raises the question: what is the evidence for a heightened autoimmune response in women that would help explain their increased susceptibility to autoimmune disease? To address this question, we examined sex differences in antigen-induced cytokine-secreting cells between untreated relapsing-remitting multiple sclerosis (RRMS) patients and controls. We studied Th1 (IFNg and TNFa) and Th2 (IL-5 and IL-10) cytokine responses to MS-relevant epitopes of myelin (MBP, PLP, MOG) as well as irrelevant epitopes. We observed significant female skewing (P V 0.005) in the IFNg response to 3 PLP peptides: PLP 40-60, [103] [104] [105] [106] [107] [108] [109] [110] [111] [112] [113] [114] [115] [116] [117] [118] [119] [120] [195] [196] [197] [198] [199] [200] [201] [202] [203] [204] [205] [206] showed a simultaneous male skewing (PV0.007) in IL-5 responses. For PLP 40-60, this resulted in a IFNg/IL-5 ratio of 174.4 in MS females, whereas the MS male IFNg/IL-5 ratio was only 0.3. MBP responses also showed strong gender interactions: MS females had very strong MBP-IFNg responses and virtually no MBP IL-5 response (P = 0.004) while the reverse was true for MS males: high IL-5 and extremely low IFNg responses (P = 0.0023). For MOG 64-96, MS females gave an IFNg/IL-5 ratio of 82.9, whereas MS males gave a ratio of 1.0. In contrast, mitogen-stimulation gave IFNg/IL-5 ratios with less than a 2 fold difference between MS females and MS males. Control females showed slightly elevated IFNg responses compared to control males, however, IFNg/IL-5 ratios were b9. These results show that MS-relevant myelin proteins can induce female MS patientsT lymphocytes to secrete inflammatory cytokines, whereas the very same myelin epitopes induce males to secrete anti-inflammatory cytokines. These interactions suggest that disease and gender are not independent factors in the immune response, but rather they interact to promote gender bias in cytokine responses that may explain why women are more susceptible to MS. A gender bias in cytokine responses could have implications for designing clinical trials involving antigenspecific therapies in MS. We have developed an approach to test a new hypothesis of the intrathymic pathogenesis of myasthenia gravis. The hypothesis posits that acetylcholine receptor alpha subunit (AChRa)specific CD4+ T cells, which escape central deletion, migrate to the thymus where, during the course of a nonspecific inflammatory reaction, they are activated by intrathymically expressed autoantigen. B6 AChRa CD4+ T cells see an immunodominant T-AChRa peptide that does not cross-react with mouse AChRa. Thus, this epitope can only be encountered in the B6 mouse thymus if we express T-AChRa there. However, it is widely regarded that 1) membrane expression of AChRs requires the proper assembly and folding of constituent subunits and 2) T-AChR subunits are assembled and expressed only at below 26 8C. We reasoned that even though T-AChRa would not assemble with mouse subunits at 37 C, it was possible that a small amount of T-AChRa could be expressed on the surface of mammalian cells in vivo at murine body temperature. We transiently transfected the tsA201cell line with Ta/pRBG4, a mammalian vector that expresses T-AChRa cDNA. In additional experiments, tsA201 cells were transfected with either 1) mouse AChRa alone, 2) T-AChRa + mouse AChRh, AChRy, and AChRe, or 3) mouse AChR a + mouse AChRh, AChRy, and AChRe (positive control). Expression of AChRa protein was investigated by flow cytometry using mAB 210, a rat IgG mAB that sees an epitope on the extracellular domain of Torpedo as well as mammalian AChRa, followed by FITC-goat anti-rat IgG antibody to detect expression of the of the transfected protein. We also used FITCa-bungarotoxin as an independent marker for detecting the alpha subunit. Mock-transfected cells served as negative controls. We observed 1) a low, albeit statistically significant, number of viable cells that had surface staining of mAB 210 after transfection with only mouse AChRa relative to mock transfected cells, 2) a similar magnitude of T-AChRa expression on cells transfected only with T-AChRa, 3) no augmentation of T-AChRa expression when the mouse AChRh, AChRy, and AChRe subunits were cotransfected and 4) many of the positive control cells (transfected with murine AChR a, h, y, and e subunits) expressing mouse AChRa. Transfected cells that were permeabilized by treatment with saponin/paraformaldehyde expressed considerable amounts of T-AChRa intracellularly. Thus, despite the temperature requirements unique to the assembly of T-AChR subunits and their membrane expression, a small amount of T-AChRa, like mouse AChRa, can be transported to the cell membrane where it is expressed as a single entity. A greater amount of the unassembled T-AChRa or mouse AChRa subunits was detected intracyoplasmically. These results indicate that unassembled T-AChRa can indeed be expressed on/in mammalian cells in vivo. They lay the groundwork for determining whether T-AChRaspecific B6 CD4+ T cells can be activated when they encounter their cognate autoantigen in the thymus in a context that promotes activation. The Cytokine Pattern of Glatiramer Acetate Reactive CD8+ T Cell Lines Derived from MS Patients and Healthy Volunteers. A. Dressel, 1 A. Vogelgesang, 1 S. Peters, 1 F. Weber. 2 1 Department of Neurology, University Greifswald, Greifswald, Germany; 2 Section of Neurology, Max-Planck-Institute of Psychiatry, Munich, Germany.Background: Multiple Sclerosis (MS) is the most frequent disabling neurological disease in young adults. Glatiramer Acetate (GA) is a synthetic amino acid copolymer that has been shown to reduce the relapse rate in patients with relapsing-remitting MS. The proposed mechanism of action of GA is functional suppression of autoreactive T cells specific for myelin antigens. GA reactive CD4+ T-cells have been generated from MS-patients and controls by us and others. As CD8+ T cells may be involved in the pathogenesis of MS, we established an experimental system to generate human, GA reactive CD8+ T-cell lines and clones, which demonstrated an antigen specific, dose dependent proliferation und IFN gamma production. The aim of our current study was to investigate the cytokine profile of GA-reactive CD8+ T cell lines derived from MS patients and controls. Results: So far 88 CD8+ T cell lines from healthy volunteers, 61 lines from GA treated MS patients and 47 lines from untreated MS patients were generated. GA reactive CD8+ T cells of untreated MS patients produced more IFN gamma (P b 0.05) and IL-4 (P b 0.001), but less IL-5 (P b 0,01) than CD8+ T cells derived from healthy volunteers. Cells derived from GA treated MS patients showed less TNF alpha (P b 0.001), lower levels of IL-10 (P b 0.001), less IL-4 (P b 0.001), but more IL-5 (P b 0.05) production than T cell lines generated from untreated MS patients. Conclusions: The data presented here demonstrate that the cytokine spectrum secreted by GA reactive CD8+ T cells differs in untreated MS patients and healthy controls and that this cytokine shift may be partly corrected by initiation of GA treatment in patients suffering from RRMS.Acknowledgments: This work was in part supported by TEVA Pharma Deutschland GmbH to FW and AD and from the Gesellschaft fqr Nervenheilkunde Mecklenburg-Vorpommern to AV and AD Multiple sclerosis (MS) is a chronic disease of the central nervous system characterized by inflammation and areas of demyelination. The role of the innate immune system in this disease is emerging. One critical family of molecules in innate immunity is the Toll-like receptor (TLR) family which plays a fundamental role in pathogen recognition and activation of the innate immune response. The TLRs have been implicated in several autoimmune diseases, but their role in MS remains unclear. We present an initial exploration of the role of two of these molecules, TLR2 and TLR4, using a retrospective study of immunophenotypes captured by the MS Natural History Database at the Partners MS Center in Boston. We analyzed data from forty-four (44) patients with MS by McDonald criteria; each subject had data from three different visits to the MS Center over the course of one year. At each visit, peripheral blood mononuclear cells (PBMCs) were collected and stained using monoclonal antibodies against CD14, TLR2 and TLR4. The expression of these molecules on PBMCs was measured both ex vivo and after stimulation with lipopolysaccharide (LPS) and ionomycin. These data were then correlated with the associated clinical phenotypes that are available in the database, including disease subtype, course, and activity as well as MRI volumetric data. Large interindividual and intraindividual variability in TLR2 and TLR4 expression on CD14+ cells was observed despite the precision of such measurements in our clinical laboratory. The role of TLR2 and TLR4 on CD14+ cells in MS remains unclear. However, these data demonstrate that TLR2 and TLR4 expression on CD14+ cells may undergo large intraindividual fluctuation over time, a fact that needs to be taken into account in any future association of these molecules with human disease. Matrix metalloproteinases (MMPs) are a family of 22 proteases, including 6 membrane-bound MMPs (MT-MMPs). Their physiological inhibitors are tissue inhibitor of metalloproteinases (TIMPs). MMPs are thought to mediate cellular infiltration in CNS inflammation, which is an integral part of the pathogenesis of Multiple Sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). MMPs may also mediate infiltration, and possibly repair, after CNS injury. We have investigated the differential expression of selected MMPs in spinal cord from mice with adoptively transferred EAE, and in a model of CNS axonal injury in the hippocampus, using real-time RT-PCR to profile changes in expression. It is highly up-regulated in EAE, where it is expressed almost exclusively by infiltrating macrophages. After axonal transection, MMP-12 expression correlated with kinetics of macrophage infiltration measured by flow cytometry in the lesion-reactive hippocampus. In CC chemokine receptor-2 (CCR-2) deficient mice, macrophage infiltration is absent. Consistently, there was no up-regulation of MMP-12 in the lesion-reactive hippocampus of CCR-2 deficient mice.After axonal transection, TIMP-1 was up-regulated prior to leukocyte infiltration of the lesion-reactive hippocampus, implicating an endogenous source. TIMP-1 may be expressed to counteract potential damaging effects of MMPs. Whereas the majority of secreted MMPs were up-regulated in EAE, 4 of 6 MT-MMPs were down-regulated. Conversely, none of the 3 MT-MMPs investigated so far were affected in the lesionreactive hippocampus. Activated microglia in the lesion-reactive hippocampus may differentially regulate MMP expression, possibly because axonal transection leads to activation of microglia in the absence of inflammation.This work was funded by the MS Society of Canada and a CIHR-IHRT grant. Searching for Biomarkers in Multiple Sclerosis: The Need for Natural History Studies. Multiple sclerosis (MS) is a chronic autoimmune disease whose main pathophysiological features are lymphocyte infiltration and inflammation, demyelination, and axonal damage of the central nervous system. This disease is heterogeneous in its clinical manifestation, prognosis, and response to different treatments. Although several therapies are available to treat patients with MS, tools to predict the course of the disease or the success of a particular treatment are still missing. The combination of different therapeutic strategies directed at the different pathophysiologic processes of the disease might be necessary. In order to achieve this goal we need to develop reliable biomarkers of disease activity and response to treatment. In this study we examined immunological biomarkers by analyzing relevant molecules on peripheral blood mononuclear cells (PBMC) from patients with MS, both ex vivo and after in vitro stimulation using flow cytometry-based assays. We measured a battery of cytokines, chemokines and their receptors, activation markers, and costimulatory molecules longitudinally in more than one hundred patients with MS enrolled in the Multiple Sclerosis Natural History Study at the Partners MS Center in Boston. The patients in different categories of disease and in different treatment subgroups were recruited and followed prospectively for up to three years with consecutive measurements of immunological markers, clinical assessment and MRI measures. Changes in biomarkers were correlated with clinical and MRI measures and with response to treatment. Our preliminary analysis shows a differential effect of various therapies on different immunological targets, some increasing anti-inflammatory response while others dramatically reducing pro-inflammatory responses at different time points after initiating therapy.This prospective multi-parameter analysis of immune markers with clinical responses and MRI support represents a systematic approach for the identification of surrogate markers of disease activity and treatment response in patients with MS. PURPOSE: Iodoform is metabolized by the cytochrome P-450 oxidative system to CO and CO 2 . In vivo, the anti-inflammatory effects of CO have been previously demonstrated in LPS-induced shock, graft rejection, and lung injury models. In EAE, increased CO has a variety of potentially beneficial effects including the protection from 1) nitric oxide synthase, 2) cellular infiltration, 3) macrophage activation, and 4) inflammatory processes including TNF-a production. Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), was chosen to study the effect of this compound in acute inflammatory autoimmune encephalomyelitis.METHODS: EAE was induced in Lewis rats after immunization on day 0 with myelin basic protein (MBP) from guinea pig. On day 10, the rats developed an inflammatory encephalomyelitis with lymphocyte infiltration and subsequent progressive paralysis. Rats injected with MBP were randomized to receive by vehicle or iodoform (250 mg/kg/day) by oral gavage. Daily clinical scores were determined on a 0-5 scale. Brain and spinal cord samples were obtained at early and peak clinical signs from the vehicle control group and corresponding treated group for cytokine and histological analysis. In addition, pharmacokinetics and blood chemistries were also determined.RESULTS: Iodoform reduced the severity of EAE in a dose dependant fashion. Significant reduction in severity was evident after 5 days of therapy (P = 0.001). Iodoform ameliorated pathological damage as determined by image analysis. Carboxyhemoglobin levels peaked within a few hours of administration but normalized within 24 hours without cumulative pharmacokinetic consequences. Analysis of brain cytokine levels indicated that iodoform increased IL-10 in EAE animals with no effect on TNF-a or IFN-g.CONCLUSIONS: These results demonstrate a powerful COmediated amelioration of EAE and suggest that further study of halothanes would be warranted to define their potential benefits in the treatment of human autoimmune diseases such as MS. Inflammation is an important aspect of autoimmune disease that contributes to the pathology of conditions such as multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). The processes that underlie inflammation in the CNS, including migration of cells and release of their effector molecules, are co-ordinated by a network of cytokines and chemokines released by resident brain cells as well as infiltrating immune cells. Following induction of EAE, mice that lack IFNg or its receptor develop a very severe disease that progresses rapidly to paralysis and death, compared to a milder relapsing-remitting phenotype in wild-type littermates. The severe disease observed in IFNg-deficient mice is characterized by lesions in the spinal cord and brainstem that are larger and more diffuse than in wild-type mice. Furthermore, inflammatory CNS infiltrates consisted primarily of macrophages/ microglia and T cells in wild-type mice, but included large numbers of polymorphonuclear cells (mainly neutrophils) in IFNg-deficient mice. It is not clear why IFNg deficiency results in more neutrophils infiltrating the CNS. We hypothesized that this is due to a change in the cytokine networks. Two possible candidate molecules are IL-17 and IL-18, both of which promote neutrophil migration and activation. We induced EAE in wild-type and IFNg-deficient mice by active immunization with PLP139-151 peptide in CFA, and investigated expression of these two cytokines by real-time PCR. IL-17 mRNA was not detectable in the spinal cord of unimmunized animals, but was induced with onset of EAE. IL-17 mRNA was expressed at significantly higher levels in the spinal cords of IFNgdeficient mice compared to wild-type mice. IL-18 mRNA and protein was expressed constitutively in the spinal cord of unimmunized mice and did not change with onset of EAE. We also investigated expression of IL-18 binding protein (IL-18BP), an endogenous, potent inhibitor of IL-18 that is regulated by IFNgamma. IL-18BP mRNA was dramatically up-regulated after onset of EAE in the spinal cord of wild-type but not IFNg-deficient mice. This study demonstrates that IFNgamma deficiency could result in aberrant IL-18 actions due to a failure to up-regulate IL-18BP. Thus, the enhanced neutrophil infiltration could be due to enhanced IL-17 expression or a lack of inhibition of IL-18. Elucidation of these pathways will help understand how perturbations of the cytokine network can impact on cellular infiltration and CNS pathology. Background: Myelin/oligodendrocyte glycoprotein (MOG) is a potent encephalitogenic antigen in experimental allergic encephalomyelitis (EAE), the animal model of multiple sclerosis (MS); it is a known target for pathogenic demyelinating antibody responses in EAE. In humans, anti-MOG antibodies have been described as prognostic markers in early MS. However, anti-MOG antibodies are not specific and can be found in a high proportion of healthy individuals, when analyzed by different methods.Objective: To validate a novel, easy to perform liquid-phase based immune assay for the detection of serum anti-MOG antibodies and to assess the different immunological properties of MOG in solution-and in solid-phase immune assays.Design/Methods: Sera of 37 MS patients with different disease stages, and 13 healthy control subjects (HC), chosen according to their reactivity against recombinant extracellular human MOG (rhMOG 125 ) determined by solid-phase ELISA, were tested for the presence of anti-rhMOG 125 IgG by incubation with the biotinylated rhMOG 125 in solution. Immunecomplexes were captured by platebound Protein G and detected by peroxidase-labeled streptavidin. A panel of monoclonal fab fragments (Fabs) and sera of 15 nonhuman primates C. jacchus marmoset immunized with either human whole white matter (HWM), recombinant ratMOG or linear 20-mer MOG peptides (n = 5 per group) were used to validate the assay.Results: No reactivity against solution-phase rhMOG 125 was detected in humans, in contrast to high anti-TT reactivity. In ELISA sera reacted equally against rhMOG 125 and TT. For anti-TT reactivity the correlation between LP and ELISA was highly significant (P b 0.001, PearsonTs correlation), validating the assay technically.In the marmoset EAE model, sera of MOG peptide immune animals reacted significantly weaker to solution-phase rhMOG 125 as compared to sera of ratMOG-or HWM-immune marmosets (mean binding ratio 1.9 vs. 33.9 (ratMOG-immune) and 14.3 (HWM-immune), respectively; P b 0.001, SNK T-test). The binding ratios of ratMOG-and HWM-immune sera were indistinguishable from each other in solution-or solid-phase assays. By means of monoclonal antibodies and fab fragments, we were able to detect a 7-32 fold lower antibody affinity against soluble rhMOG 125 compared to solid-phase rhMOG 125 .Conclusions: We conclude that (1) The inflammatory myopathies are putative autoimmune disorders characterized by muscle weakness and the presence of inflammatory infiltrates in skeletal muscle. In inclusion body myositis (IBM), the inflammatory cells that are infiltrating muscle have been characterized as predominately CD8+ cytotoxic T lymphocytes. While there does not appear to be significant B cell infiltrates as indicated by CD20 immunohistochemistry, we recently demonstrated the presence of significant muscle infiltrates of CD138+ plasma cells. Here, we examined the immunoglobulin variable region sequences of these cells. B cell immunoglobulin variable region gene libraries of IBM muscle were created by reverse transcriptase-polymerase chain reaction followed by sequencing of the whole immunoglobulin cDNA variable region, allowing identification of somatic mutations. There was significant oligoclonal expansion found in IBM muscle, in contrast to libraries from peripheral blood mononuclear cells that showed no oligoclonal expansion. Moreover, a pattern of B cell affinity maturation was evident in that clones sharing the same CDR3 had accumulated varying numbers of common and unique somatic mutations within the CDR regions and this clonal variation was also observed within groups comprised of CD19+ and CD138+ B cells. These data suggest that a local inflammatory response occurs within the muscle tissue of patients with IBM that may indicate the presence of a humoral antigen-specific response present in muscle. Interest in examining the genetic nature of single cells has been shared by investigators in different fields. The limited amount of genetic material from a single cell specimen presents a technical challenge even for the most efficient and specific PCR protocols.In the present report we show the application of a recently developed method for whole genome amplification (WGA), the multiple displacement amplification (MDA) (Dean, Gen Res 2001), to single cells FACS-sorted from human blood. This procedure allows the interrogation of a single cellTs genetic information through the production of several copies of the single cells genome followed by specific PCR experiments on a small fraction of the MDA product. We also compare MDA to whole genome amplification of single cells using the primer extension pre-amplification (PEP) method.This technique can be applied to the analysis of the clonality and antigen-specificity of T-cells and B-cells which infiltrate tissues in autoimmune diseases. In fact, it allows the sequencing of both chains of immunoglobulin and T-cell receptor V-(D)-J rearranged genes from single cells isolated from frozen human tissue. Single cells are dissected from the stained tissue with the use of a laser capture microdissection device, their DNA is amplified with MDA, then a small fraction of the amplification product is used as template for the specific PCRs. Furthermore, the combination of laser capture microdissection and MDA allows to study single cells/cell types from paraffin-embedded tissues, where the RNA is mostly degraded but the DNA remains intact. The notion that the process of B cell affinity maturation with ensuing production of potentially pathogenic autoantibodies may occur inside the CNS of patients with multiple sclerosis (MS) is supported by the presence, within lesions, of oligoclonal B cells and cell surface markers capable of supporting such a local immune response. Although B cells carrying somatic mutations of Ig variable (V) region genes have been detected in the CSF and CNS tissue of patients with MS, more direct evidence for the process of B cell affinity maturation confined within this compartment is needed. Here, we have characterized the B cell Ig variable (V) region genes derived from multiple lesions within the white matter of patients with MS. Analysis of variable region gene libraries revealed evidence of significant oligoclonal expansion of local B and plasma cells. Moreover, a pattern of B cell affinity maturation was evident in that clones sharing the same CDR3 had accumulated varying numbers of common and unique somatic mutations within the CDR regions and this clonal variation was also observed within groups comprised of CD19+ and CD138+ B cells. These data suggest that a local inflammatory response occurs within the CNS tissue of patients with MS which includes differentiation and affinity maturation. Auto-or exogenous antigens resident in the CNS need to be considered as driving such a response. SJL mice are highly susceptible to the induction of experimental autoimmune encephalomyelitis (EAE) with myelin proteolipid protein (PLP) peptide 139-151, whereas H-2 congenic B10.S mice are resistant. Immunodominance and susceptibility to PLP 139-151induced EAE was found to be associated with a high precursor frequency of PLP 139-151-specific T cells in the naive repertoire of SJL mice. To understand the mechanism for disease resistance in B10.S mice, we determined the precursor frequency of PLP 139-151-reactive T cells in both strains of mice using IAs / PLP 139-151 tetramers. Both SJL and B10.S mice had similar frequencies of tetramer-reactive T cells in the naRve peripheral repertoire. However, in SJL mice the majority of PLP 139-151 tetramer-positive cells were in the CD4+CD25-population whereas there were more tetramer-positive cells in the CD4+CD25+ population of B10.S mice, suggesting that there were more PLP 139-151-specific T cells in regulatory population. Depletion of CD4+CD25+ cells in vivo facilitated expansion of PLP 139-151-reactive cells with production of TH1 cytokines in EAE-resistant B10.S mice. Furthermore, depletion of CD25+ T cells with anti-CD25 antibody treatment prior to immunization with the encephalitogenic peptide resulted in induction of EAE in these otherwise resistant mice. These data indicate a role for autoantigen-specific CD4+CD25+ cells in genetic resistance to autoimmunity. A 20 year-old female with Systemic Onset Juvenile Idiopathic Arthritis (SoJIA)presents to University Hospital with right knee arthritis and pharyngitis previously treated with naproxen and penicillin with no response. Vancomycin and Ceftriaxone were started after bilateral arthrocenteses (each appearing inflammatory) were performed. Following the second arthrocentesis she experienced right shoulder and pleuritic chest pain. Naproxen and prednisone were started. Labs showed: CRP 196 mg/dL; normal liver and renal functions. HIV, ANA, double stranded DNA, RPR and RF were negative. New cervical and inguinal adenopathy, splenomegaly, abdominal distention and right upper quadrant pain were noted. A light erythematous, nonpruritic, macular rash covered her anterior neck and shoulders. Over three days acute renal failure and liver function abnormalities developed; non-steroidals were discontinued. Bilateral infiltrates evolved on her chest radiograph. On HD #11 she had a generalized tonic-clonic seizure, was intubated and transferred to the intensive care unit (ICU). The next few days were marked by hypotension, tachycardia and persistent fevers (N101 o F). She developed clonus, asymmetric plantar reflexes and obtundation. Despite her condition, ESR was only 5, but CRP was 24 and ferritin was 152,197. Macrophage Activation Syndrome (MAS), a complication of SoJIA was considered and high dose solumedrol and cyclosporin were started. Blood product support, hemodialysis and pressor management were instituted. She was discharged home on HD #31 on a prednisone taper and cyclosporin. Bone marrow biopsy during her ICU course showed prominent hemophagocytosis. TNFa, IL2 and IL6 levels were markedly elevated during her critical illness, but normalized by the time of discharge. MAS is a secondary hemophagocytic syndrome most commonly associated with SoJIA. Its symptoms are attributed to activation and proliferation of well-differentiated macrophages precipitated by a change in medication or infectious cause. Presenting symptoms include pyrexia, changes in mentation, organomegaly, lymphadenopathy, bleeding, bruising and purpura; paradoxically arthritis and serositis can improve. Laboratory abnormalities can include pancytopenia, transaminase elevation, coagulopathy, decreased ESR (hypofibrinogenemia), hypertriglyceridemia, hyponatremia, hypoalbuminemia and hyperferritenemia. Histological evaluation of bone marrow shows macrophage hemophagocytosis. Mortality exceeds 20%, but with rapid diagnosis and the institution of high dose steroids, cyclosporin and other immunomodulators, good outcomes can be achieved. Background: Following activation of antigen-presenting cells (APCs), co-stimulatory pathways including B7-1 and B7-2 recognizing CD28 and CTLA-4 play a key role in the activation of autorreactive lymphocytes, also CD40 ligand is expressed by activated T cells and is considered to be a critically T-cell marker, and their increased expression might further contribute to cognate T-B cell interactions and autoantibody production. To succeed an appropriate interaction between T and B cells is required an appropriate signaling of co-stimulatory molecules, being the broadly studied; B7-CD28 and B7-CTL4 who are crucial in both pathways IL-2 production and tolerance induction. Objective: The aim of this study was to explode, which preferably co-stimulatory via in APCs interacting with CD4+ T cells, and which are the correlation with activity disease in Systemic Lupus Erythematosus and Rheumatoid Arthritis patients. The proportion of peripheral mononuclear cells was studied using flow cytometry in order to measure the percentage of surface molecules in CD4+ T cells, and antigen presenting cells such CD14+ and CD19+. Results: CD4+ CD152+ T cells showed a high expression only in Lupus patients, CD80+ and CD86+ in both type of cells CD14+ and CD19+ showed increased levels of those molecules predominantly in Lupus patients.Conclusion: Costimulatory molecules play an essential role in the activation and regulation of T cell immune responses in lupus and arthritis patients trough CD28, CD30, CD152, CD154, CD80 and CD86, they could be used to monitoring the activity disease and also could be a target for therapeutic manipulation of the costimulatory system in order to beneficial effects in clinical autoimmune disease Keywords: PBMCs, CD4+, CD28+, CD14+, CD19+, CD152, CD154, co-stimulatory molecules, CD80, CD86, disease activity, Systemic Lupus Erythematosus and Rheumatoid Arthritis. Therapy, Chiba, Chiba, Japan; 4 Med. Univ., Kanazawa, Isikawa, Japan.The extract from Taxus yunnanensis (TY) had many beneficial effects in certain clinical settings, in which physicians can easily use on patients without interrupting therapy. We will show the long-term effect of TY on patients with severe pain due to rheumatoid arthritis (RA). After prescribing the TY extract, the extent of morning stiffness, joint swelling and pain were reduced, and the lowered patientsT quality of life (due to severe pain caused by RA) was significantly improved with increasing daily life activities. However, the beneficial effects of TY did not appear shortly, although TY had an immediate relieving effect on allergic diseases. Plasma cytokine levels were not decreased promptly. Although walking ability of the patients got better after 3 days, it took around 8-20 days and 1 month to improve morning stiffness and joint pain, respectively. Therefore, a longer prescribing period seems to be necessary to demonstrate the beneficial effect of TY. The following two patients were good responders to TY; they were prescribed TY extract in addition to the usual therapeutics for RA. It is noteworthy that their plasma IL-6 levels reduced to the normal level after taking TY.Case 1 (Class 4 disability): Plasma IL-6 level dramatically reduced from 165pg/ml to the normal level 32 weeks after prescribing TY. RF also became negative at 20 weeks.Case 2 (Class 3 disability): Plasma IL-6 decreased from 6.8pg/ ml to the normal level 16 weeks after prescribing TY. RF activity in the serum improved from 220 to 78U/ml. complement and adaptive T cell and B cell immune responses. Upon binding of its ligand C3d, CR2 lowers the threshold for B cell activation; however, the function of CR2 on T cells is unknown. Mice deficient in CR2 and CR1 (Cr2-/-) demonstrate altered humoral immunity in response to foreign and self antigens as well as an altered natural antibody repertoire. Collagen-induced arthritis (CIA), a model of autoimmune arthritis, depends upon complement activation, effective collagen presentation by antigen presenting cells, autoantibody production by B cells, and cytokine production by T cells. Therefore, CIA provides a model in which the roles of lineage-specific expression of CR2 and CR1 in autoimmune disease may be dissected. DBA/1j mice were immunized with bovine type II collagen (CII) emulsified in complete FreundTs adjuvant (CFA) on days 0 and 21 to establish CIA. On day 35, draining lymph nodes from mice with CIA demonstrated a greater percentage of CD4+ cells expressing CR2 compared to naRve mice (3.59% vs. 1.29%, P = 0.005, respectively), suggesting a role for CR2 expression on T cells during autoimmune disease.To determine the importance of CR2/CR1 for the development of CIA, Cr2-/-and Cr2 F mice backcrossed 5 generations onto the DBA/1j strain were generated. Mice were immunized with CII in CFA on days 0 and 21 and evaluated in a blinded fashion for the development of arthritis. Cr2-/-mice (n = 20) had significantly reduced severity (2.5 F 0.9 vs. 5.5 F 1.2, P = 0.05) and incidence (45% vs. 64%) of arthritis compared to Cr2 F mice (n = 25). However, anti-bovine and anti-murine CII antibodies did not differ significantly between the two groups of mice, nor did cellular proliferation and cytokine production in response to CII restimulation ex vivo differ between the two groups. C3 deposition within the joints of Cr2-/-mice was significantly reduced, suggesting that activation of complement in these mice was altered. These results demonstrate that CR2 and CR1 are required for robust development of CIA, likely because of altered T cell activation and trafficking to the joint or because of defects within the anti-CII antibody repertoire. Leptin is an adipocytokine that links the metabolic status to several important immune functions. We and others have recently shown that leptin, similarly to other pro-inflammatory cytokines, can promote the differentiation of T helper (Th1) cells and can contribute to the onset and progression of organ-specific autoimmunity in several animal models of autoimmune disease. Nonetheless, the role of leptin in systemic autoimmunity, and in particular in systemic lupus erythematosus (SLE), remains elusive. We studied here whether leptin exerted some influence on the development and progression of systemic autoimmunity in lupusprone (NZB Â NZW)F 1 (BWF1) mice. We found by ELISA that that the circulating levels of serum leptin increased progressively with age in untreated female mice ( P b 0.0002 at 13 and 20 weeks vs 1 week of age). Importantly, treatment of BWF1 mice with recombinant leptin promoted Th1 autoreactivity (as indicated by predominant IgG2a rather than IgG1 anti-double stranded (ds)DNA antibody responses). Histological studies indicated that leptin accelerated production of autoantibodies and favored deposition of immune complexes and kidney glomerular damage in the mice treated with leptin, as compared to saline-treated control mice. Interestingly, intraperitoneal injection of a single dose of 100 Ag of anti-leptin antibodies to severely nephritic mice (proteinuria z 300 mg/dl) delayed progression of renal disease and significantly prolonged survival of the treated mice (P b 0.001 by the Mann-Whitney U test at six weeks posttreatment). Taken together, these studies point to an important role of leptin in the development and progression of lupus in BWF1 mice and raise the possibility to consider leptin antagonists as novel therapeutic tools for immune intervention in systemic autoimmunity. Hyperhomocysteinemia is a risk factor for trombosis and recurrent pregnancy loss. Similar mechanisms appear to mediate thrombosis and pregnancy loss associated with hyperhomocysteinemia and with antiphospholipid antibodies. To report on the case of a patient with the association of hyperhomocysteinemia and antiphospholipid or Hughes syndrome; and to assess the prevalence of increased levels of homocysteine in a group of patients with Hughes syndrome. Materials and Results. A 41-year old woman with a previous history of migraine, placental abruption, arterial hipertension, unstable angina and multinodular goitre was admitted because of two consecutive transitory ischemic attacks. Laboratory investigations revealed the presence of elevated titres of anticardiolipin antibodies (66 GPL-units/ml) and significant hyperhomocysteinemia [homocysteine concentration, 36 umol/L (normal values: 1.6-11 umol/L)]. The genotype of the termolabile C677T allele of 5,10 methylene tetrahydrofolate reductase was studied by polimerase chain reaction. The patient was found to be a carrier of the C677T homozygous genotype. Hyperhomocysteinemia was normalized. We performed a retrospective study on 23 patients with clinical and laboratory features of the Hughes syndrome in comparison with 42 patients with unexplained fetal loss (without antiphospholipid antibodies) and with 51 patients with thrombotic events (without antiphospholipid antibodies). Rates of hyperhomocysteinemia were statistically similar in patients with Hughes syndrome and in disease control groups (17.4%, 9.5% and 23.5%, respectively). Using a cut-off point of 11 umol/l, four patients with Hughes syndrome had hyperhomocysteinemia. Three patients had thrombotic events (including the presented case) and the other patient had 9 consecutive unexplained abortions. Homocysteine levels were similar in patients with Hughes Syndrome and disease controls (10 F 0.6, 6.9 F 0.5 and 9.98 F 0.8 umol/l, respectively). Patients with thrombosis (without antiphospholipid antibodies) had significantly higher levels of plasma homocysteine than women with abortions (P = 0.018). The results presented in this study suggest that hyperhomocysteinemia is not significantly increased in patients with Hughes syndrome. However, antiphospholipid antibodies may coexist with high levels of plasma homocysteine in individual cases of Hughes Syndrome. Atherosclerosis in Murine SLE. Zhongjie Ma, 1 Marc Monestier, 2 Robert Eisenberg. 1 1 Department of Medicine, University of Pennsylvania, Philadelphia, PA; 2 Department of Microbiology and Immunology, Temple University, Philadelphia, PA.Introduction: The accelerated development of atherosclerosis and increased risk of cardiovascular disease in young women with systemic lupus erythematosus (SLE) is a disturbing feature of the disease that is not well understood. We have combined mouse models of SLE and atherosclerosis to begin to elucidate the mechanisms of this disease synergy.Methods: Chronic graft-versus-host (cGVH) disease was induced in young apoEKO C57BL/6 mice by injection of 10E8 coisogenic bm12 spleen cells. Mice were maintained on normal chow diet. Mice were sacrificed, and the hearts and aorta were collected at 16 weeks after induction of cGVH for histology. The cholesterol levels were measured with an enzymatic colorimetric method. The frozen heart tissues were stained with Oil red O and hematoxylin, and the aortas were stained with Sudan IV. En face lesion areas were calculated with image-pro 5.0 software system. Serum IgG, anti-dsDNA, anti-chromatin, anti-oxLDL, and anticardiolipin levels were measured by ELISA. Proteinuria was detected with Uristix reagent strips at sixteen weeks after induction of GVH. Spleen cells were stained with immunofluorescence and detected with FACS.Results: The plasma cholesterol levels in the apoEKO mice were greatly increased, and this was not significantly changed by cGVH. ApoEKO mice with and without cGVH had substantial lesions in the aortic root and aorta tree, as well as some lesions in the coronary arteries, which were slightly increased by cGVH. ApoEKO mice had increased numbers of splenic marginal zone B cells, which were depleted by cGVH.Conclusion: These results indicate that we can induce cGVH and lupus-like autoimmunity in apoEKOmice, and alter some of the manifestations associated with Atherosclerotic cardiovascular disease (ASCVD). We have extended this approach to other mouse lupus models, such as C57BL/6-lpr/lpr. Objective: It has been demonstrated previously that a single nucleotide polymorphism C1858T in the protein tyrosine phosphatase gene PTPN22 is associated with a rheumatoid factor (RF) positive subset of patients with known rheumatoid arthritis. Our study was performed to investigate the association between this polymorphism and the presence of RF in a healthy population.Methods: Healthy subjects were recruited in Denver, Colorado as part of the ongoing Studies of the Etiologies of Rheumatoid Arthritis (SERA) project. At the time of this interim analysis, 334 subjects were available [mean age of 38 (range 29-56), 88% non-Hispanic white, and 69% female]. Each of these subjects provided epidemiologic information and underwent an interview and physical examination to ensure that no subjects included in the analysis had evidence of RA. Serum samples were drawn and the presence of RF was determined using nephelometry. The PTPN22 polymorphism (1858C -N T) was identified using MGB-Eclipsek Probe System (Epoch Biosciences, Inc), performed at the Benaroya Research Institute, Seattle, Washington. Statistical analysis was performed using logistic regression (SAS version 8) .Results: In this healthy population, 45 out of 334 (13%) subjects had a positive RF. After adjusting for age, gender, race, smoking status, and shared epitope status, the PTPN22 polymorphism was marginally associated with the presence RF (OR 2.02, Confidence Limits[CL] 0.92-4.45), P = 0.08).Conclusions: In this preliminary analysis of healthy subjects, the 1858C -N T missense single nucleotide polymorphism in PTPN22 is marginally associated with the presence of RF. The size of the odds ratio is similar to that reported previously for this polymorphismTs association with RF positive RA patients and will likely become statistically significant once we complete patient accrual (estimated 450 subjects by Spring 2006). This gene may contribute to pre-clinical immune dysregulation and the initial development of RA-specific autoimmunity. Anti Histone antibodies were positive in 8 out of 19 (mean titers: 98.5 F 61.9 U/ml)) available SLN sera (42%) while 6 out of 7 (85.7%) OLN showed augmented serum levels (mean titers: 84.5 F 62.8). Anti dsDNA were elevated in 28/29 (96.5%) SLN patients(mean titers: 98.7 F 88.3 U/ml) and in 10/11 (91%) of OLN individuals (mean titers: 54.2 F 30.7 U/ml). Prevalence and mean titers of these three autoantibodies were significantly different than those encountered in 25 control sera (P b 0.01). In addition in SLN patients, serum levels of Anti C1q and Anti Histone antibodies significantly correlated with Anti dsDNA levels and with increased activity index in renal tissue (P b 0.05). In those SLN with detectable Anti C1q antibodies, IgG (66%), C1q (44%), C3 (77%) and C4 (55%) deposits were found.Conclusions: This is the first report of detectable Anti C1q and Anti Histone autoantibodies in SLN. Furthermore, their significant correlation with Anti dsDNA antibodies and with increased activity index in renal tissue suggest an early and simultaneous participation of these complexes in Lupus Nephritis. Introduction: Granulocyte apheresis (GCAP) is a novel hemoadsorption treatment that is currently being used for the treatment of some autoimmune diseases. Inmunomodulatory properties of GCAP have been reported associated to emerging evidence of clinical improvement in patients.Objective: To assess the efficacy and safety of AdacolumnR GCAP in patients with refractory rheumatoid arthritis (RA).Methods: Patients with active RA who had failed to respond to at least one DMARDs or biologics (TNF-alpha antagonists) were treated with weekly GCAP for five weeks. Clinical assessments and response to therapy were analyzed at weeks 5,7,12 and 20 in an open multicenter pilot trial. The primary outcome measure of therapeutic response was the 20% improvement in the American College of Rheumatology criteria at week 20. EULAR response criteria based in the disease activity score for 28 joints (DAS-28) and disability by the Healt Assessment Questionnaire (HAQ) were also analyzed.Results: Twenty seven patients were enrolled: 81.5% were women with mean disease duration of 14.4 years. The mean number of previous DMARDs was 3.7 and 48.1% of them have failed to biologics. On an intent to treat basis analysis 40.7% of patients achieved an ACR20 improvement and 44.4% patients a therapeutic EULAR response at week 20. These percentages were of 50% and 54.5% in the 22 patients who completed the trial. In four out of the 10 patients who completed the trial and previously failed to biologics, an ACR20 response was achieved at week 20. A significant decrease was noted in the different ACR response components, including the tender joint count, swollen joint count, pain score and the patientTs and physicianTs global assessment and also the DAS28 index; most of them improve since week 5. The treatment was well tolerated and only one serious adverse event related to study therapy was documented (sepsis due to catheter infection).Conclusions: Treatment with GCAP led to significant clinical improvement in a subset of patients with RA who previously failed to DMARDs or biologics. The therapy was safe and well tolerated. Further large, placebo controlled studies are required to assess the exact role of this therapy in refractory RA. Gene expression studies have demonstrated increased interferon (IFN)-inducible gene (IFIG) expression in peripheral blood mononuclear cells (PBMC) of many patients with systemic lupus erythematosus (SLE). Our recent data have implicated a predominant type I IFN effect in the IFIG expression observed in SLE. The objective of this study was to examine the hypothesis that increased disease severity and activity as well distinct autoantibody specificities characterize SLE patients with type I IFN pathway activation. In order to do that, freshly isolated PBMC from 77 SLE patients, 22 disease controls (DC), and 28 healthy donors (HD) were subjected to real-time PCR for 3 IFIG that are preferentially induced by IFNa, and the data were used to derive IFNa scores for all individuals. Expression of IFIG was significantly higher in SLE patients compared to DC or HD. SLE patients with high (H) and low (L) IFNa scores were compared for clinical manifestations of disease, disease severity, disease activity, serologic features and potential confounders by bivariate and multivariate analysis. We found that SLE patients with a H IFNa score had significantly higher prevalence of renal disease, a greater number of ACR criteria for SLE, and a higher SLICC damage index (DI) score than SLE patients with L IFNa scores. Patients with H scores showed increased disease activity, as measured by lower C3, hemoglobin, absolute lymphocyte count, and albumin, and higher anti-dsDNA titer, ESR, and SLEDAI-2K score. The presence of antibodies specific for RNA-binding proteins (RBP: Ro, U1-RNP, Sm), and dsDNA, but not phospholipids, was significantly associated with a H IFNa score. Logistic regression analysis confirmed that renal disease, higher SLICC DI scores, low complement levels, and presence of anti-RNA binding protein (RBP) autoantibodies were independently associated with a H IFNa score, and suggested that the same might be true for the absence of treatment with hydroxychloroquine (HCQ). In conclusion, activation of the IFNa pathway defines a subgroup of SLE patients characterized by increased disease severity, including renal disease, increased serologic disease activity, and autoreactivity to RBP. These data provide support for the further examination of the role of IFNa score as a potential biomarker for lupus disease activity and suggest a pathogenic link between RBP and IFNa production.Sa1.57. The Rheumatic Joint Contains Hyperreactive CD28 null CD4 + T Cells.A. A subpopulation of unusual CD4 + T cells lacking the costimulatory molecule CD28 can be found in a subpopulation of patients with chronic inflammation and to a lesser extent in healthy individuals. These CD28 null CD4 + T cells are potent secretors of TNF and IFN-g, and proliferate vigorously upon stimulation. We, and others, have previously demonstrated that circulating CD28 null CD4 + T cells can constitute of up to 50% of CD4 + T cells in peripheral blood (PB) from patients with chronic rheumatic diseases.Aim. Are CD28 null CD4 + T cells present also in the inflamed joint of rheumatic patients? Mononuclear cells were isolated from PB and synovial fluid (SF), from inflamed knee joints, of patients with different rheumatic diseases. The cells were sorted into CD28 null and conventional CD28 + CD4 + T cells and stimulated in vitro by anti-CD3 without the presence of antigen presenting cells.Results. CD28 null CD4 + T cells could be found in synovial fluid from patients, but only in individuals displaying this population also in their peripheral blood. These CD28-negative cells from the inflamed joints were confirmed to be CD28 null cells since they had the same restricted TCR Vbeta repertoire as the corresponding cells in the circulation. The joint derived CD28 null cells were as proliferative and prone to secrete proinflammatory cytokines as the CD28 null cells in PB.Conclusion. When present in the joint CD28 null cells is likely to contribute to the inflammation as they easily expand and secrete proinflammatory cytokines. Ongoing efforts include investigations of synovial tissue for CD28 null cells and linking the presence of these hyperreactive T cells in the joint to a clinical feature.Sa1.58. Absence of B Cells Decreases Both Proliferation and Cytokine Secretion.The aim is to analyze if the reduced proliferation and cytokine secretion of mononuclear cells in systemic lupus erytematosus (SLE) patients treated with the B cell depleting therapy Rituximab can be explained by the absence of B cells.Background and methods: Rituximab, an anti CD20 antibody therapy, was originally developed against lymphomas, but is now increasingly used in autoimmune diseases. Our earlier studies of Rituximab treated SLE patients have shown that shortly after treatment both proliferation and cytokine secretion decreased in in vitro cultures, and increased with the return of B cells in the circulation. To investigate if it is the lack of B cells or the immunosupressive treatment given together with Rituximab that accounts for this dramatic effect, we established an in vitro system. B cells were depleted by anti CD20 magnetic beads from peripheral blood mononuclear cells (PBMC) from three healthy subjects. Cells were stimulated with PHA or anti-CD3 antibodies, with or without anti-CD28 co-stimulation. Cytokine secretion and proliferation were measured with cytometric bead array and 3 H-Thymidine incorporation.Results: Similar to our ex vivo patient data, both proliferation and cytokine secretion were reduced in B cell depleted PBMC cultures as compared to intact PBMC cultures. This was true for both PHA and anti-CD3 stimulated cells.Mainly TNFa, IL-10 and IL-6, but also IL-4 and IL-2, were secreted to a lesser extent.Discussion: Our in vitro experiments indicate that it is not the immunosuppressive treatment that accounts for the decreased immune response seen in Rituximab treated SLE patients, but a per se effect of the lacking B cells. Future experiments will delineate if it is their potential to co-stimulate, to present antigens, or to secrete cytokines that is most important for the activation of T cells. Natural killer T cells (NKT) are a population of regulatory T cells that co-express an invariant T cell receptor as well as NK cell markers. Several studies have shown that NKT cells are decreased or dysfunctional in autoimmune conditions such as insulindependent diabetes mellitus, systemic sclerosis, systemic lupus erythematosus and multiple sclerosis. Significant therapeutic effects of a-GalactosylCeramide (a-GalCer), a synthetic antigen of NKT cells, have been demonstrated in animal models of autoimmunity. NKT cells have therefore been implicated to participate in the regulatory immune mechanisms controlling autoimmunity. However, their role in the pathogenesis of rheumatoid arthritis (RA) remains unclear. To this end, we studied the frequency, cytokine profile and heterogeneity of NKT cells in peripheral blood mononuclear cells (PBMC) of 23 RA patients and 22 healthy controls, which included paired PBMC-synovial fluid (SF) samples of 7 and paired PBMC-synovial tissue (ST) samples of 4 RA patients, respectively. Using flow cytometry, a decreased NKT cell frequency was observed in blood of RA patients compared to healthy controls. In addition, direct ex vivo ELISPOT analysis revealed a reduced IL-4/IFN-g ratio in NKT cells of RA patients. The invariant T cell receptor sequence was detected in paired SF and ST samples. NKT cells of all healthy controls, but only of 53.8% of the RA patients (responders) expanded upon in vitro stimulation. However, reactivity towards a-GalCer was observed in NKT cells isolated from SF of both responder and non-responder RA patients. Intracellular FACS analysis of the cytokine profile of CD4+ and CD4-PBMC derived NKT cell lines of RA patients revealed that both produced significantly less IL-4 compared to those of healthy controls. In contrast, SF derived NKT cell lines displayed a Th0 phenotype comparable to that of healthy controls. These findings suggest that SF NKT cells are functional, even in patients with non-responding NKT cells in the blood.In conclusion, our data demonstrate that NKT cells are decreased and biased towards a Th1 phenotype in blood, but are not impaired in SF of RA patients. This indicates NKT cells that might be functionally related to resistance or progression of rheumatoid arthritis. Neutralizing agents to TNF are the most successful means to ameliorate systemic autoimmune inflammation. Neutralization of TNF, however, is often associated with the development of autoantibodies, in particular to nuclear antigens, the mechanisms of which are unknown. Here, we analyzed the effect of TNF and its neutralization on MHC class II expression and function of antigen presenting myeloid cells in rheumatoid arthritis (RA). Monocytes were isolated from the peripheral blood of RA patients before and after anti-TNF-mAb treatment and from controls by negative selection, differentiated in vitro into macrophages and analyzed by flowcytometry for HLA-DR expression. T cell responses to activation by myeloid cells were assessed in proliferation assays, and mRNA levels of the class II transactivator (CIITA) were determined by semiquantitative RT-PCR. HLA-DR expression was significantly reduced on myeloid cells from RA patients with active disease, but was increased to normal levels after TNF mAb treatment. Concordantly, in vitro application of TNF to monocytes from healthy individuals reduced their ability to upregulate HLA-DR during differentiation to macrophages and, importantly, inhibited their ability to stimulate T cells in mixed lymphocyte reactions. The data indicate that TNF decreases HLA-DR expression by reducing CIITA mRNA levels in myeloid cells, functionally resulting in a decreased stimulatory capacity of myeloid cells for T cells. Concordantly, ameliorating disease activity in chronic inflammatory diseases by neutralizing TNF restores HLA-DR expression of myeloid cells and their ability to stimulate T cells. Thus, anti-TNF treatment might lead to augmented T cell activation by myeloid cells, thereby promoting immune responses to (auto)antigens and the development of antinuclear antibodies that are frequently associated with anti-TNF therapy.Sa1.61. Anti-mitochondrial (AMA) of M5 type antibodies were initially described in patients with autoimmune diseases, and then have been identified in sera of patients with antiphospholipid antibodies and recurrent foetal loss, haemolytic anaemia and thrombocytopaenia, and have controversial association with thrombosis. Up to date, very scarce literature exists regarding M5 type AMA. AMA of M5 type are directed towards an unknown antigen located in the inner membranes of mitochondria (50 kDa). Indeed, M5 type AMA have been always reported in the context of antiphospholipid syndrome strictly linked to antiphospholipid antibodies.We report here on a 65-years-old Caucasian woman diagnosed as autoimmune polyglandular syndrome (APS) IIIC type, namely autoimmune thyroiditis, pernicious anaemia and recurrent idiopathic thrombocytopaenic purpura. Clinical history was also relevant for two foetal losses at 2 and 4 gestational months, respectively, associated to persistent M5 type AMA at high titre (1/640). Antinuclear, anti-DNA and antiphospholipid antibodies (anticardiolipin, anti-beta-2-glicoprotein-I) were all of them persistently negative through a 10-years follow-up period. Coagulation studies were repeatedly normal.Conclusion: In our patient, type 5 AMA was the only marker of thrombocytopenia and recurrent miscarriages without antiphospholipid antibodies. In isolated cases, M5 Abs appear to be a diagnostic marker for clinical manifestations of antiphospholipid syndrome. Background Natural regulatory T cells are usually identified in two ways; by mRNA expression of the transcription factor FOXP3 or by the level of surface expression of CD25. Humans are never immunologically naRve, resulting in T cells expressing CD25 also due to activation. Thus, frequency determinations based on CD25 expressing regulatory T cells can never be more than rough estimates. Objective In this study we investigate the presence of regulatory T cells in different compartments of patients with rheumatic joint disease. We compare CD25 and FOXP3 in peripheral blood and the site of inflammation, by analyzing both synovial fluid and synovial tissue.Materials and methods FOXP3 mRNA levels were investigated from sorted peripheral blood and synovial fluid CD4 T cells populations. The sorted populations expressed different densities of CD25. RNA was also prepared from synovial tissue biopsies. Additionally, CD25-T cells were activated in vitro to investigate possible induction of FOXP3.Results and discussion We could find FOXP3 message in all compartments investigated, even in biopsies from synovial tissue. In synovial fluid, the CD25bright cells were markedly enriched for FOXP3 compared to the CD25int, while the difference between the two CD25 populations were less apparent in blood. Interestingly, also cells negative for CD25 could be FOXP3+, and this was more common in synovial fluid than in blood.Thus, our study shows that FOXP3+ regulatory T cells are not restricted solely to CD25+ T cells. Especially in an inflammatory environment like synovial fluid FOXP3+ CD25-T cells were found. An induction of FOXP3 in CD25-cells could not be mimicked in vitro perhaps suggesting that these regulatory T cells originate from CD25+ cells that have downregulated or shed their CD25 expression. Such a scenario is supported by studies showing the presence of soluble CD25 in both sera and synovial fluid from rheumatic patients. Objective: An abnormal host defense against pathogens is implicated in the pathogenesis of spondyloarthropathy (SpA), a disease characterized by abundant synovial infiltration with innate immune cells. Considering the role of Toll-like receptors (TLRs) in activation of innate inflammation and occurence of TLR-dependent infections after TNFalpha blockade, we analyzed TLRs in SpA and their modulation by TNFalpha blockade.Methods: Peripheral blood monocytes were obtained in SpA and rheumatoid arthritis (RA) during infliximab therapy and in healthy controls (HC). Expression of TLR2 and TLR4 and TNFalpha production upon LPS stimulation were analyzed by flowcytometry on different monocyte subsets. Synovial biopsies from 23 SpA before and after infliximab or etanercept treatment and from 15 RA were analyzed by immunohistochemistry.Results: TLR4, but not TLR2, expression was increased on monocytes in SpA, whereas both TLRs were increased in RA. The CD163+ macrophage subset, which is increased at the inflammatory sites in SpA, has a particularly increased TLR expression. Accordingly, expression of both TLRs was significantly higher in SpA than in RA synovium. Infliximab decreased TLR2 and TLR4 expression on monocytes in SpA and RA, leading to lower levels than in HC and to an impaired TNFalpha production upon LPS stimulation. Paralleling the systemic effect, synovial TLRs were downregulated following infliximab as well as etanercept, indicating a class-effect of TNFalpha blockers.Conclusions: SpA inflammation is characterized by increased TLR2 and TLR4 expression which are sharply reduced by TNFalpha blockade. These data emphasize a central role for innate immune-mediated inflammation in SpA and provide an additional clue for the efficacy as well as the potential side-effects of TNFalpha blockade. Background: Previously, we demonstrated anti-nuclear antibody (ANA) and anti-dsDNA antibody induction after 30/34 weeks of infliximab therapy in rheumatoid arthritis (RA) and spondyloarthropathy (SpA).Aim: To further assess in detail the clinical and biological correlates of autoantibody induction during longer-term TNFalpha blockade with either the monoclonal antibody infliximab or the soluble receptor etanercept.Methods: 34 SpA and 59 RA patients were treated with infliximab for two years. Additionally, 20 SpA patients were treated with etanercept for one year, providing a unique head-tohead comparison of autoantibody induction during TNFalpha blockade in a human disease model with low baseline autoimmunity. Sera were blindly analysed for ANA, anti-dsDNA, anti-ENA, anti-histone and anti-cardiolipin antibodies. The anti-dsDNA antibodies were further isotyped with gamma-, mu-and alphachain specific conjugates.Results: In the infliximab-treated SpA and RA cohorts, we observed high numbers of newly induced ANA (61.8% and 40.7%) and anti-dsDNA antibodies (70.6% and 49.2%) after one year, but no further increase between year 1 and year 2. In contrast, induction of ANA (10%) or anti-dsDNA antibodies (10%) was only occasionally found in the etanercept-treated SpA cohort. Neither during infliximab nor etanercept, anti-ENA, anti-histone antibodies or clinically relevant lupus-like symptoms were described. Similarly, infliximab but not etanercept selectively increased the IgM but not the IgG anti-cardiolipin titers.Conclusion: This study indicates that the prominent ANA and anti-dsDNA autoantibody response is not a pure class effect of TNFalpha blockers, is independent of the disease background and is not associated with clinically relevant lupus-symptoms. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease and immune function in SLE is paradoxically characterized by active T cell help for autoantibody production, along with impaired T cell proliferative and cytokine responses in vitro. Recently, evidence reveals that chemokines as well as chemokine receptors are closely involved in initiating the hyperreactivity. This study was designed to investigate the expression levels of various chemokines and their receptors in relation to the disease activity of juvenile SLE, and to compare the pattern of chemokine elevations with that in normal individuals. Serum levels of chemokines including CCL-2, CCL-5, CXCL-8, -9 and-10 were analyzed by chemokine cytomeric beads arrays (CBA), whereas the expression of chemokine receptors such as CCR-2, -3, -4, and-5 on peripheral blood mononuclear cells (PBMC) were assessed by real-time RT-PCR and/or Western blot. Here we demonstrate the difference of chemokines and their receptors expression in juvenile SLE patients compared with normal individuals. Significantly higher serum levels of CCL-2 (MCP-1), CXCL-8 (IL-8), -9 (MIG) and-10 (IP-10) were found in most SLE patients analyzed, while their chemokine receptors such as CCR-2, -3, -4, and-5 were expressed relatively low in patientsT PBMC. Analysis between clinical manifestations such as SLEDAI (Systemic Lupus Erythematosus Disease Activity Index) and levels of the above chemokines expression levels revealed a strong correlation. However, decreased serum levels of CCL-2, CXCL-9 and CXCL-10 appeared in patients with high levels of anti-dsDNA if compared with those in patients with low anti-dsDNA. Overall, four chemokines that were elevated in SLE were proinflammatory, characteristic of activation of the monocyte and macrophage lineage, and in the case of IL-8, also of neutrophils. These data suggest a major role for a cellmediated immune response occurring in the pathophysiology of SLE.Sa1.66. Modulation of Murine Lupus by an Inhibitory GpG Oligonucleotide.K. 2 1 Department of Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, CA, USA; 2 Department of Neurology, Stanford University School of Medicine, Stanford, CA, USA.Activation of the innate immune system by DNA containing hypomethylated CpG motifs has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). We have previously described an immunomodulatory oligodeoxynucleotide (ODN), containing a single base switch from CpG to GpG, which ameliorated murine experimental autoimmune encephalomyelitis (EAE), a T helper 1-mediated model of human multiple sclerosis. Here, we examined the consequences of immunostimulatory CpG-ODN and inhibitory GpG-ODN treatment in the NZBÂNZW F 1 (NZB/W) murine model of SLE. Beginning at 5 months of age, we administered CpG or GpG ODNs at regular intervals to female NZB/W animals, over a period of 20 weeks. While CpG-ODN treatment did not appear to impact overall disease severity, GpG-ODN treatment significantly delayed the onset of proteinuria, and improved 40-week survival in NZB/W mice. We also determined the effects of ODN administration on NZB/W T lymphocyte cytokine profiles and splenocyte surface marker expression. Interestingly, GpG-ODN treatment enhanced production of TNF-a by NZB/W T cells, and inhibited T cell production of IL-4. Consistent with observations made in the EAE model, CD11c + splenocytes derived from GpG-ODN treated NZB/W mice also displayed reduced surface expression of CD80 and CD86. Taken together, the data indicate that GpG-ODN treatment can modulate immune cell function and ameliorate disease in the NZB/W model of lupus nephritis. The protective mechanism of the GpG-ODN in murine SLE may involve general inhibitory effects on costimulatory molecule expression by antigen presenting cells, as well as alteration of T cell cytokine profiles. Clinical trials of the oral treatment of patients with autoimmune diseases including rheumatoid arthritis and multiple sclerosis with type II collagen and myelin, respectively, showed disappointing results. This may be in part due to the insufficient induction of oral tolerance to the respective autoantigen in humans. Therefore, we have looked for agents that can facilitate induction of oral tolerance. In the present study, we tested the hypothesis that the phosphodiesterase IV inhibitor rolipram, that was previously reported to produce suppressive cytokines including TGF-beta and IL-10, can facilitate the suppression of antigen-induced arthritis (AIA) in mice by oral administration of the inducing antigen. Such suppressive cytokines have been shown to play a role in oral tolerance, especially induced by low doses of oral antigen. To prove the hypothesis, DBA/1J mice were immunized with ovalbumin (OVA) emulsified with CFA (day 0). Oral tolerance was induced by oral administration of either 0.1 or 10 mg of OVA daily over a period of 5 consecutive days commencing on day -5. The results showed that oral administration of 0.1 and 10 mg of OVA alone was followed by suppression of AIA, although the extent of suppression of AIA was greater in mice fed 10 than 0.1 mg of the oral antigen. When 0.1 mg of OVA was given together with rolipram, significantly facilitated suppression of AIA was observed. Secretion of IFN-gamma from spleen cells was suppressed by 0.1mg of oral OVA alone and this suppression was significantly enhanced in mice given both the antigen and rolipram. There was no difference in the secretion of IL-10 between either 0.1 or 10 mg of OVA alone-and OVA plus rolipram-treated groups. This may be in part explained by significantly accelerated decreases in IFN-gamma in mice treated with the lose dose of OVA plus rolipram. In our studies, the facilitated suppression of AIA by the administration of the oral antigen together with the phosphodiesterase IV inhibitor does not appear to be mediated by the modulation of IL-10 secretion. Agents such as rolipram that facilitate induction of oral tolerance might be useful in the treatment of autoimmune diseases in humans including rheumatoid arthritis with oral pathogenic autoantigens. Objective: Rituximab, an anti-CD20 monoclonal antibody, has become a target for immunotherapy of B cell lymphomas and, more recently, B cell-mediated autoimmune diseases. We report a case involving a 45 year old female patient with severe autoimmune disease and B cell immunodeficiency who was treated with rituximab.Findings: The patient initially presented at 42 years of age with RaynaudTs phenomenon, positive anti-nuclear antibody (ANA), and positive thyroid peroxidase antibodies. Fever, oral and vaginal ulcers, polyarthritis, digital vasculitis, elevated erythrocyte sedimentation rate, and elevated rheumatoid factor (RF) also developed. A classic dermatomyositis rash was confirmed by skin biopsy. Subsequently, her amyopathic dermatomyositis was treated with intravenous immunoglobulin (IVIG) 25 grams in addition to methotrexate 25 mg weekly and cyclosporin resulting in limited improvement. Upon our immune evaluation, lymphocyte studies showed a low B cell percentage of 4%, a low absolute B cell count of 53 cells/ml, increased CD4+CD45RA+ naRve T cells at 51%, and low CD4CD45RO+ memory T cells at 27%. Additionally, lymphocytic mitogenic responses were markedly decreased to StaphA, a B cell mitogen. Also, there was an increase in C3D immune complexes. RF was elevated at 58 IU/ml and Epstein Barr Virus (EBV) serology showed a high titer viral capsid antigen IgG and high titer early antigen antibody suggestive of reactivated EBV disease. Treatment of dermatomyositis with an underlying B cell immunodeficiency was started with high dose IVIG at 1 gram/kg of Gammunex combined with cyclophosphamide and steroids. Thereafter, all in vitro markers of autoimmunity including C-reactive protein, ANA, direct Coombs, and RF normalized. However, her pulmonary and upper extremity vasculitis progressed. Rituximab therapy was considered because our immune evaluation revealed a preponderance of CD20+ cells. Five weekly doses of rituximab at 375 mg/m 2 were added to the high-dose IVIG, cyclophosphamide, and steroid therapy. The pulmonary vasculitis improved, digital infarcts and ulcers slowly healed, and all autoimmune markers including RF became normal. Immune studies repeatedly showed b1% CD19+ and CD20+ cells at 24 weeks. The patient was weaned off cyclophosphamide and remains on IVIG and low dose prednisone with no further exacerbations of her autoimmune disease at 24 weeks.Conclusion: Our patient with a severe, refractory autoimmune disease and underlying B cell defect responded successfully to addition of rituximab, specifically targeting a B-cell mediated autoimmune process. The favorable response of rituximab in our patient is supported by recent published reports showing B cell depletion with rituximab led to a sustained clinical response in methotrexate-resistant rheumatoid arthritis. Purpose: To determine if several of the DMARDs, B-blockers, ACE-I, or aspirin are associated with the presence of coronary calcification by Electron Beam Computed Tomography (EBCT) in SLE patients.Methods: One hundred and thirty seven patients with SLE over the age of 18 who fulfilled at least 4 of the American College of Rheumatology criteria for the classification of SLE were recruited for the study. A history, physical exam, EKG and EBCT measuring coronary calcium were performed. Results from the EBCT were used as an independent measure of current cardiovascular disease. The information regarding current, past and number of years on a medication was recorded. Analysis inculding standard studentTs ttest was performed on the data.Results: A t-test analysis of the data showed that when comparing patients with SLE who were currently on azathioprine to patients with SLE who were not currently taking the drug, those on the drug had a lower EBCT calcium score, (P = 0.03). Results were unchanged when beverQ users were added to the analysis. Comparison of SLE patients taking hydroxycholoroquine to those who were never on hydroxycholoroquine showed no difference in EBCT calcium score, (P = 0.59). Comparison of SLE patients on prednisone to those SLE patients never on prednisone showed no difference in EBCT score, (P = 0.35). SLE patients having received intravenous cyclophosphamide also showed no difference in EBCT score compared to those who were never exposed to the drug, (P = 0.48). SLE patients taking mycophenolate mofetil and methotrexate similarly showed no difference in EBCT score for those exposed versus not exposed, (P = 0.15 and P = 0.24 respectively). SLE patients on statins had a higher EBCT calcium score than those not on the drugs, (P = 0.02) and SLE patients on B-blockers also had a higher EBCT score than those not on the drug, (P = .008). Patients on ACE-I and aspirin show no difference in EBCT score compared to those not on these drugs, (P = 0.24 and P = 0.22, respectively).Discussion: Ever or current azathioprine use is associated with lower EBCT calcium score in SLE patients. The other DMARDs studied were not associated with lower coronary calcification. The higher EBCT score associated with B-blocker and statin use is likely associated with previously identified cardiovascular risk factors in these patients. Rheumatoid arthritis (RA) is an autoimmune synovitis characterized by heterogeneity between patients in disease manifesta-tions, clinical outcomes and therapeutic responses. We applied synovial antigen microarrays and a bead-based multiplex cytokine assay to profile autoantibody and cytokine responses in RA, with the objective of identifying profiles of secreted proteins in blood that provide diagnostic information and delineate disease subtypes. We demonstrate that autoreactive B cell responses targeting deiminated epitopes and elevated serum concentrations of proinflammatory cytokines (TNFa, IL-1a, IL-1b, IL-6, and IL-15) were present in a subset of early RA patients with features predictive for development of severe RA. In contrast, autoimmune targeting of the native epitopes contained on synovial arrays, including human glycoprotein 39 and collagen types II and V, and low concentrations of serum cytokines were associated with predictors of lesssevere RA. Ligation of Death Receptor 5 (DR5) induces cell death in activated B and T cells. Thus, DR5 is an attractive target for therapeutic elimination of pathogenic lymphocytes in autoimmune diseases. To determine which lymphocytes express DR5, subsets of B and T cells from human blood, tonsil and spleen were identified by surface markers and analyzed for binding of anti-DR5 mAb. Germinal center B cells in the tonsil and spleen expressed DR5, as did plasmablasts in these tissues and in blood. CD38intermediate cells in the blood did not, consistent with the phenotypic characterization of these as transitional cells. The increase in plasmablasts in the circulation in SLE subjects resulted in an increase in the percentage of blood B cells expressing DR5. A small but reproducible increase in DR5 expression on post-naive subsets was observed in both CD4 and CD8 T cells in healthy subjects. However, DR5 expression was significantly greater in these same subsets of T cells from lupus subjects, compared to the same subsets in healthy controls. Thus, DR5 expression appears to be modulated by both differentiation and by other factors, possibly including disease-associated factors such as type 1 IFN. Therapeutic targeting of DR5-expressing cells would spare resting B cells and naive T cells but has the potential to eliminate activated cells to a degree that would be determined by disease-specific mechanisms. Background/Purpose: Anti-citrulline antibodies are highly specific serologic markers for RA and the immune response to citrulline is linked to the expression of the RA shared epitope. While citrullination of polypeptides under the influence of peptidyl arginine deiminase (PAD) has been shown to be unregulated in normal mice following the induction of inflamma-tion (Streptococcal cell wall (SCW) induced), these mice do not develop an immune response to citrulline or chronic arthritis. We sought to investigate the arthritogenic properties of and immune response to citrullinated proteins following acute SCW arthritis induction in DR4-tg mice.Methods: 25 Ag of streptococcal cell wall antigen (Streptococcus pyogenes Group A PGPS 10S, Lee Laboratories Grayson, Georgia, USA) in 5.0 Al of PBS was injected into one knee joint of DR4 tg and wt mice. The other knee joint of these mice received 5.0 Al of PBS. Mice were sacrificed at various time points to investigate pathological changes, and T and B-cell immune responses.Results: Pathology of the injected joint from day 2 demonstrated a massive influx of leukocytes and the start of synovial proliferation. At day 7, synovial hyperplasia, the first signs of erosion at the bone cartilage surface by the proliferating synoviocytes, and depletion of approximately 50% of the proteoglycan normally present in the articular cartilage was observed. Splenic T-cell proliferation was observed in the DR4 tg mice at various time points with a citrullinated peptide of the a chain of fibrinogen (QDF TNCit INK LKN S) but this was not evident in the wt mice.No T-cell proliferation was detected in either group of mice when stimulated with the unmodified version of this peptide.Conclusion: SCW as expected induced an acute inflammatory arthritis in both normal wt and DR4 tg mice. This was followed by a strong citrulline specific T-cell response only in the DR4 tg mice, presumably by the inflammatory induced expression of PAD and citrullinated fibrinogen. These observations are consistent with our previous studies indicating that the T-cell response to citrulline is restricted by MHC class II molecules expressing the shared epitope. This young girl, with antecedents of recurrent otitis media, was admitted to the hospital for a polyarticular arthritis with fever, significant inflammatory syndrome (increased ESR, C-reactive protein and fibrinogen), mild normocytic anaemia, abnormal liver function tests but no eye inflammation. She was found to have antinuclear antibodies at 1:640 on Hep2 cells with a speckled pattern and nuclear dots. Antibodies against extractable nuclear antigens and nDNA were not found. Complement analyses showed the absence of serum hemolytic activity, normal C4, elevated C3 levels and undetectable C2. This complotype was linked on one chromosome to the typical HLA-A*25, B*18, DRB1*15 (2), DQB1*06(1) bancestralQ haplotype but on the other chromosome to HLA-A*2, B*27, DRB1*13(6), DQB1*06(1) which represents a very unusual association with the S042 complotype. Upon serum immunoglobulin determinations, IgD and IgG4 immunoglobulins could not be detected, IgA and IgM levels were close to the lower normal range. Altogether, the clinical syndrome in this patient was related both to homozygous C2 deficiency and positivity for HLA-B27. Although deficiencies in the components of the classical pathway of complement activation were among the first identified risk factors for systemic lupus erythematosus (SLE), only a few studies addressed their significance in patients with cutaneous lupus erythematosus (CLE). Among environmental factors, it was postulated that cigarette smoking might intervene in the pathogenesis of LE.In a retrospective study of 85 patients with CLE, 32 individuals were screened for C4 and/or C2 deficiency. Among them 17 had a C4A deficiency (1 homozygous-16 heterozygous), five a C4B deficiency (2 homozygous-3 heterozygous), and two a combined heterozygous C2 and C4A deficiency. The serum level of C4 was decreased in 40 % of patients with C4B deficiency and in only 5 % of patients with C4A deficiency. A high proportion (58 %) of these complement-deficient patients were male (F/M ratio = 0.70); the mean age at diagnosis was 36 years. Of particular interest was the detection of a combined heterozygous C2 (type I) and heterozygous C4A deficiency in two male patients. In this series, 82 % of the patients were smokers and 94 % of male patients with CLE were smokers. It has been recently suggested that smoking behaviour could be related to specific major histocompatibility complex haplotype(s) on chromosome 6 characterized by the presence of a C4A null allele. 1 Internal Medicine and Rheumatology, Juntendo University School of Medicine, Tokyo, Japan.Objective: Only a few studies on pregnancy outcome in patients with mixed connective tissue disease(MCTD) are available and their results are contradictory. The purpose of this study is to examine pregnancy and fetal outcomes in MCTD patients.Methods: A retrospective study we have followed ten mothers with MCTD, during their pregnancies since 1999 to 2003 at Juntendo University Hospital.Result: 1. Of the 10 pregnancies, we observed 5(50%) live birth at term, 2(20%) premature birth, 2(20%) spontaneous abortion, 1(10%) artificial abortion.2. One case had interstitial pneumonia and high titer of sialyl carbohydrate antigen KL-6 following no therapy, KL-6 level was decreased during pregnancy. At 37 weeks of gestation, the laboratory findings were suggestive of early HELLP syndrome, and the pregnancy was terminated.3. Serum anti-phospholipid antibodies were not determined either. And active nephritis was recognized at the pregnancy.Conclusions: Since MCTD has a relatively good prognosis, no therapy changes are required during pregnancy. But premature delivery and spontaneous abortion were high rate in this study. It considered that organic involvement and flare are possibility of risk for pregnancy with MCTD women. Characteristics of Patients with Clinical Manifestations of APS with Anti-beta 2 -Glycoprotein-I but Not Anticardiolipin Antibodies or Any Other Autoimmune Condition. Fli1 also has been implicated in the regulation of the immune system and autoimmunity. Fli1 is expressed in the thymus and spleen and its overexpression in mice results in the development of immunological renal disease similar to that observed in systemic lupus erythematosus. Elevated expression of Fli1 has been observed in the spleen of lupus mouse models NZB/NZW f1 and MRL/lpr. Furthermore, elevated levels of Fli1 in peripheral blood monocytes from lupus patients correlates with disease activity. Interestingly, MRL/lpr mice that have a heterozygous knockout of Fli1, and a subsequent 50% reduction in Fli1 expression, have significantly reduced renal disease and prolonged survival. To further understand the role of Fli1 in the immune system we are examining the regulation of Fli1 in normal and lupus mice. Our preliminary results show that Fli1 expression is higher in CD19+ and CD8+ cells from predisease NZM2410 and MRL/lpr lupus prone mice and in CD8+ cells from late disease MRL/lpr mice compared to BALB/c mice. Transient transfections of Fli1 promoter/reporter constructs into a B cell line indicate that the highest level of expression is driven by a 400 bp region encompassing most of exon 1 and that most of the positive regulatory elements necessary for this expression are localized within a 200 bp region. Furthermore, we have examined the promoter and upstream regulatory regions of Fli1 from BALB/c, NZM2410 and MRL/lpr spleen and identified a polymorphism in exon 1. Transient transfection analyses indicate this polymorphic region contributes to the positive regulation of Fli1 promoter/reporter constructs. Interestingly, this region is highly homologous to the human Fli1 sequence and is located adjacent to a regulatory element found to be necessary for Fli1 expression in a leukemia cell line. Further analyses of the regulatory region, including the polymorphism, will allow further insight into what elements and binding factors are necessary for normal expression, as well as aberrant expression in lupus prone mice. Exposure of breeder mice to vinyl chloride has been shown to result in proliferation of microchimeric cells and parallel development of skin fibrosis. Herein, we describe immunological findings in a male patient with lymphocytic infiltrates and fibrosis involving skin, lungs and the digestive tract highly reminiscent of cGVHD, following exposure to organic solvents. We provide evidence that maternal microchimerism could be involved in the disease process.The patient was lymphopenic with a CD4/CD8 ratio of 0.33. Lymphocyte phenotyping revealed a high proportion of circulating T cells bearing activation markers, among both CD4 (81% CD25+, including 42% CD25high cells) and CD8 (23% CD25+) populations. Oligoclonal expansion of T cells was demonstrated by flow cytometry and immunoscope analysis, involving CD8+ cells belonging to Vh7 (32% of CD8 cells) and Vh17 (24% of CD8 cells) families. Both Vh7+CD8+ and Vh7+CD8+ cell populations stained negatively for CD27, CD28 and perforin. High resolution HLA typing of the patient and his mother demonstrated that they were nearly identical for MHC class II (both were DRB1*1104 DRB1*1302 and DQB1*0301 DQB1*0604, patient DPB1*0301 DPB1*0401, mother DPB1*0301 DPB1*0402), but not for MHC class I molecules.Search for microchimeric cells of maternal origin by FISH in peripheral blood revealed the presence of 7 XX cells among a total of 40600 analysed cells (0.017%) from the patient.Bidirectional mixed lymphocyte cultures (MLCs) were performed using T cells from the patient and irradiated non-T cells from his mother, and vice versa, to explore the potential functional consequences of maternal microchimerism in this patient. In presence of patient non-T cells, maternal CD8+ cells showed an increase of HLA-DR expression (24% vs 8.4% when cultured alone) but neither proliferation nor IFN-gamma production, whereas CD4 cells remained quiescent. In contrast, both CD4+ and CD8+ T cells derived from the patient were activated in presence of maternal non-T cells, as shown by increased HLA-DR expression (41.6% vs 16.4% in absence of maternal non-T cells for CD4+ cells, and 32% vs 11% for CD8+ cells) and secretion of IFN-gamma. Removal of patient CD4+CD25high cells from cultures resulted in decreased overall activation of patient T cells in response to maternal non-T cells, indicating that they were effector and not regulatory T cells.This observation suggests that chronic activation of T lymphocytes related to long term persistence of maternal cells and exposure to organic solvents might lead to a GVH-like disease reminiscent of SSc. These IgG anti-CII antibodies are generally pathogenic, as they can trigger joint inflammation via interactions with IgG Fc receptors (FcgR), notably FcgRIII. However, SWR mice are resistant to the arthritogenic properties of these antibodies. Considering this, we have in the present study investigated if possible FcgR polymorphisms are involved in the susceptibility to CIA. This was studied by generating mice carrying the FcgRIII gene from the arthritis susceptible DBA/1 mouse (F4.D +/+) or from the SWR mouse (F4.S +/+). After CII-immunization, F4.D +/+ mice, but not F4.S +/+ mice, developed a progressively severe arthritis. In addition, the direct effect of IgG anti-CII antibodies on arthritis development was studied by passive transfer of a cocktail of monoclonal anti-CII antibodies to F4.D +/+ and F4.S +/+ mice. Like in actively induced arthritis, F4.D +/+ mice developed a severe arthritis in contrast to F4.S +/+ mice, which were almost protected from disease. We found that FcgRIII exhibits three different haplotypes in mice, FcgRIII:V, FcgRIII:H and FcgRIII:T, and that SWR (FcgRIII:V) and DBA/1 mice (FcgRIII:H) indeed differ in the FcgRIII gene. Interestingly, the DBA/1 mouse shared the FcgRIII:H haplotype with the autoimmune-prone strains, NZW, NZB, BXSB, NOD and MRL. We also demonstrate that SWR and DBA/1 mice differ at the level of FcgRIIB, displaying the Ly-17.1 or the Ly-17.2 haplotype respectively. These results suggest that polymorphisms in FcgRs may form the basis of one aspect of susceptibility to autoimmune arthritis. INTRODUCTION: About 10-20% of systemic lupus erythematosus SLE develops during childhood. The aim of this study was to describe the clinical manifestations and outcomes of a national cohort of pediatric patients with SLE.PATIENTS / METHODS: We have collected retrospective data on all cases meeting the ACR diagnostic criteria of childhood onset SLE, registered in the Israeli national registry of children with rheumatic diseases, who were diagnosed and followed between 1987-2003. We examined disease activity and damage by using SLE disease activity index (SLEDAI), and SLE collaborating clinics/ACR (SLICC/ACR) disease damage.RESULTS: 102 patients were identified. 81% were females. The mean age at diagnosis was 13.3 F 2.6 years (range 6.9-17.7) Initial clinical manifestations included renal involvement in 41%, CNS in 7%, hematological in 94%, malar rash in 49%, oral or nasal ulcerations in 21%, musculoskeletal in 45%, and serositis in 16%. 80 children (80%) started therapy with corticosteroids, and 19 (19) with immunosuppressive drug. 55 (66%) were still on corticosteroids and 27 (32%) were on immunosuppressive drugs. 44 (73%) were on corticosteroids and 23 (38%) were on immunosuppressive drugs. 28 (64%) of them were on steroids, 22 (50%) on immunosuppressive drugs. Five patients developed chronic renal failure, one died.CONCLUSIONS: In our national cohort the 5-year outcome of pediatric SLE was good; the damage index was very low with relatively low activity in most patients. Forty-six lupus patients under 45 years old who fulfilled the American College of Rheumatology revised criteria for the classification of SLE were selected. The exclusion criteria consisted of: renal failure, nephrotic syndrome, thyroid and liver disease, diabetes mellitus, obesity, pregnancy, taking drugs that induce dyslipidemia. Disease activity was measured by systemic lupus erythematosus disease activity index criteria. The controls were forty-one healthy indivisuals matched for age ( F 3 years) and sex. According to the lipid profiles, in active, inactive and control groups, we found the high level of serum triglycerides and VLDL and low level of serum HDL in active group compared with inactive group (P b 0.05). This pattern of dyslipoproteinemia was observed in patients with positive anti-dsDNA antibodies when compared with patients with negative anti-dsDNA antibodies (P b 0.05). This pattern of dyslipoproteinemia in active SLE is attributable to autoimmune mechanisms especially in relation to the presence of anti-dsDNA antibodies. RNA editing is the co-or post-transcriptional modification of RNA which results in the insertion, deletion or substitution of nucleotides. RNA editing correct, extend or diversify the information encoded within the corresponding genomic sequence, and frequently alter the function of the affected RNAs. Therefore, RNA editing plays an important role in the regulation of gene expression and in the induction of phenotypic variability. The occurrence of high circulating levels of type I interferons (IFNs) in SLE has been well documented. Our previous experiments demonstrated upregulation of type I IFN inducible RNA editing gene, 150-kDa ADAR1 expression in SLE T cells. Goal of these experiments is to identify the role of type I IFN inducible ADAR1 in editing of protein kinase A (PKA) and ADAR2 gene transcripts of normal and SLE patients. cDNAs synthesized from T cells of SLE and normal control groups were amplified using PKA and ADAR specific primers. The amplified products of the PKA and ADAR2 transcripts were cloned into pCR2.1-TOPO vectors. Novel A Y G editing sites were identified in the PKA and ADAR2 gene transcripts of SLE T lymphocytes. The ADAR1 gene up-regulation suggests a possible cause for PKA and ADAR2 gene transcript editing in SLE T cells. In addition to AY G, novel T (U) Y C editing was also observed in PKA and ADAR2 gene transcripts of normal and SLE T cells. The enzyme responsible for such editing and the mechanisms underlying such editing are unknown. Taken together, these results clearly indicate the increased occurrence of mRNA editing in the PKA and ADAR2 gene transcripts of SLE T lymphocytes. Mutant gene transcripts are pathophysiologically significant, for they can encode diverse, aberrant forms, including truncated, dominantnegatives, resulting in abnormal gene function. Therefore, it is proposed that deficient and/or abnormal activity of genes such as PKA and ADAR2 will contribute to the pathogenesis of SLE by impairing T cell functions. To determine if B cells of lupus prone NZB mice possess intrinsic defects that directly lead or contribute to T cell hyperresponsiveness, we injected age, sex and MHC II matched NZB and Balb/c mice with histone peptide H471 representing a dominant Th cell epitope in histone H4 of the nucleosome. We cocultured purified CD4+ T and B220+ B cells of naRve or peptide primed NZB and Balb/c mice in the presence of the peptide. We found that B220+ B cells of NZB mice express high levels of surface CD86 following antigen priming. Antigen presentation exclusively by autoimmune B cells of NZB mice induced hyperresponsiveness from normal CD4+ T cells of Balb/ c mice. T cell hyperresponsiveness is a result of CD86 costimulation by B cells of NZB mice. Induction of nasal tolerance to H471 in NZB mice suppressed CD86 surface expression and led to downregulation of T cell proliferative response and cytokine production. More interestingly, B220+ B cells purified from nasally tolerized NZB mice induced T cell anergy to anti-CD3 and anti-CD28 antibody stimulation in vitro. The anergic T cells do not possess suppressive function in coculture with naRve T cells nor produce suppressive cytokines interleukin 10 (IL-10) and transforming growth factor-beta (TGF-b) upon anti-CD3 and anti-CD28 antibody stimulation in vitro. Synovial Fluid and Inflammatory Response in Rheumatoid Arthritis. 1 Clinic for Rheumatology and Clinical Immunology, Military Medical Academy, Belgrade, Belgrade, Serbia, Yugoslavia.The TH1 immunologic reaction is the major amplification factor in pathogeneses of the rheumatoid arthritis (RA). Rheumatoid arthritis is destructive synovitis of autoimmune nature. Cytokines TH1 lymphocytes with products of synoviocyte disrupt natural balance in cytokine network inside synovial tissue, which leads to inflammatory reaction and joint damage. (1), carrying diverse modifications at the hydroxylysine (Hyl) side chain were designed and synthesized to explore the fine specificity of bCII-reactive T cells involved in the initiation and/or regulation of collagen-induced arthritis (CIA), a mouse model for rheumatoid arthritis (RA). The required h-D-Galactosyl-(5R)-5-Hydroxy-L-lysine and corresponding mimetics conveniently protected for solid phase synthesis were all obtained by a divergent route featuring enantiopure 5-hydroxylated 6-oxo-1,2piperidinedicarboxylates as key intermediates. All three bCIIspecific T hybridomas used in this study as well as a recurrent pathogenic CD4 + T cell clone isolated from bCII-immunized DBA/1 mice recognized the galactosylated form 1 of the immunodominant bCII (256-270) epitope. These cells were extremely sensitive to changes at the q-amino group but differ in their pattern of recognition of analogues with Hyl side chain modified at C-5 (i.e. inversion of stereochemistry, methylation). These data further document the importance of collagen posttranslational modifications in autoimmunity and in the CIA model in particular and provide new insight on the molecular interaction between glycopeptide 1 and the TCR of pathogenic T cells.Sa1.86. CD8A on Monocytes May Aggravate Immune-Complex Mediated Disease by Binding MHC Class I and Enhancing TNF Production. 1 1 Medicine, University of Alberta, Edmonton, AB, Canada.CD8a is expressed by monocytes and macrophages (Mo and M) in rats, but according to available evidence not in mice. CD8+ Mo and M are present in several rat models of diseases involving immune complexes, such as glomerulonephritis, arthritis, and ischaemia. Depletion of CD8+ cells mitigates pathology in these disease models. If Mo and M express CD8, they may be partially responsible for pathology in some of these diseases in rats and humans previously ascribed to CD8+ T cells. TNF is therapeutically targeted in many of these diseases, and released in large quantity by Mo. We hypothesized CD8 on human Mo may be involved in TNF-mediated pathology in immune complex diseases. Ten to 25 percent of monocytes expressed high amounts of CD8a, while the remaining 75-90% of monocytes expressed low amounts of CD8a. A proportion of Mo and lymphocytes can be difficult to distinguish by some methods including cell morphology, flow cytometry (FSC-SSC gating), and potentially anti-CD3 mAb labelling. Moreover, because many of the anti-CD8a mAb used here are sold for clinical evaluation (e.g. OKT8, B9.11, LT8, 51.1), to avoid confusion between CD8 hi Mo with CD8 hi lymphocytes in clinical and research settings, careful definition of T cells (e.g. CD8a protein was found on the surface of a Mo cell line (THP-1) in continuous culture that also expressed CD8a mRNA. In the absence of another source of CD8a, THP-1 and likely other Mo transcribe and translate CD8a. Functionally, Mo may use both CD8a and FcgR to bind tissues containing immune complexes. We established that Mo can bind tetramers of MHC class I (HLA-A2), independent of bound peptide. CD8a accounted for some MHC class I tetramer binding to Mo, as this was partially inhibited by some anti-CD8a mAb. In agreement with literature, not all CD8a mAb blocked binding of MHC class I, demonstrating that mAb binding to the surface of Mo does not indiscriminately block binding of MHC class I tetramers. Select anti-CD8a mAb but not non-specific mAb imbedded in immune complexes enhanced Mo TNF production. Similar studies with other anti-CD8a mAb did not induce TNF production, suggesting that particular epitopes of CD8a activate Mo, and mAb specific for surface proteins of Mo do not indiscriminately enhance TNF production. The ability of Mo to bind MHC class I and release TNF through CD8a shows that Mo CD8a could be involved in initiating and aggravating pathology induced by immune-complexes. It is possible that some effects attributed to CD8+ T cells, where T cells have been inadequately characterized, are due in part to CD8+ Mo. Introduction: Several studies have shown the diagnostic usefulness of anti-cyclic citrullinated peptide (CCP) and anti-Sa antibodies in rheumatoid arthritis (RA), but up to now there is no study that has assessed both autoantibodies simultaneously in a cohort of patients.Objective: To determine the sensitivity, the specificity, the positive (PPV) and negative (NPV) predictive values of anti-CCP and anti-Sa in a monographic out-patient clinic of CICTD.Methods: Cross-sectional study. Anti-CCP and anti-Sa antibodies were identified by ELISA and immunoblotting techniques, respectively.Results: Anti-CCP antibodies were detected in 63/87 RA (sensitivity: 72,4% specifity: 94,4%, PPV: 87,5%, NPV: 81,9%), 3/19 PMR, 2 palindromic rheumatism (PR), 1 systemic lupus eritematosus, 1 undifferentiated connective tissue disease (UCTD), 1 ankylosing spondylitis (AS) and 1 undifferentiated espondyloarthritides (sensitivity 72.4%, specificity 94.4%, positive predictive value 87.5% and negative predictive value 86.5%). Anti-Sa antibodies were detected in 38/87 patients with RA (Sensitivity: 43,6%, specifity: 96,3%, PPV: 86,3%, NPV: 76,2%), 2 UCTD, 1 SjfgrenTs syndrome, 1 PR, 1 AS and 1 juvenile idiopathic arthritis.Conclusions: The specificity and the predictive values of anti-CCP and anti-Sa antibodies for the diagnosis of RA are comparable. However, the sensitivity of anti-CCP antibodies is higher, given the higher sensitivity of the ELISA technique when compared with immunoblotting. Introduction: Modified extracellular matrix (ECM) proteins such as hydrolyzed (elastin, collagen fibronectin, thrombospondin, etc.) or polymerized proteins (collagen-PVP), has been shown to regulate inflammatory processes. Due to, the evaluation of the effect in osteoarthritis (OA) and rheumatoid arthritis (RA), diseases related to chronic inflammation, results of a special interest. Material and Methods: Cartilage and synovial tissue co-cultures from 5 patients with RA (ACR) or 5 patients with OA (ACR) were performed. All of them were prescribed for total knee or hip replacement surgery. Tissues were cultured with RPMI-1640, 10% SFB, antibiotics and antimicotics during 7 days under the following conditions: a) RPMI (control), b) 1% collagen-PVP, c) 1% hydrolyzed collagen and elastin and d) 1% hydrolyzed collagen and elastin + 1% collagen-PVP. In order to determine the effect of the different culture conditions on the ECM turnover (elastin, collagen and sulfate proteoglycans and hyaluronic acid) tissues were stained with Hematoxilin and Eosin, Verhoeff and Alcian Blue staining techniques. Proinflammatory cytokines (IL-1b, IL-8, IL-10, IL-12, TNF-a, and IFN-g) in supernatants were quantified by ELISA. Data were normalized by total protein concentration evaluated by the Folin-Lowry micro-method. IL-1b, TNF-a and Ki-67 expression was determined by histochemistry. Results: The histological analysis showed a remodeling tissue, related to an increase of highly sulphated proteoglycans, sialomucin and hyaluronic acid. A scarce increment of elastin fibers in tissues treaded with the mixture of hydrolyzed and collagen-PVP vs. control cultures were observed. A 2-3-fold increment of Ki-67 was determined in the tissues treated vs. control cultures. IL-1b and TNF-a were determined 1.5-2-fold lower levels in treated cultures vs. controls. IL-8 levels were decreased in all supernatants from treated co-cultures from RA patients. However, only in supernatants from OA tissue treated with hydrolyzed proteins statistically decreased vs. the controls was determined. Conclusions: Modified proteins induce a tissue remodeling, promoting the recovery of cartilage proteoglycans, down-regulating the expression from some proinflammatory cytokines and promoting the chondroid cells proliferation. Introduction: The afodrine is localised in the plasma membrane of most mammalsT cells. After the cell break-down during apoptosis by the action of the caspase-3, a cleavage-product of the alpha-fodrin of 120 kD acts as a neoantigen, being recognised by sera of patients with SS. 90-60% of patients with SS presented positive IgA anti-afodrin antibodies.Objective: To assess the frequency and the clinical associations of IgA anti-alpha-fodrin antibodies in patients with primary and secondary SS in our environment.Materials and Methods: We studied 491 patients diagnosed of SS, 209 primary SS and 282 secondary SS: 190 rheumatoid arthritis (RA), 59 systemic lupus eritematosus (LES), 13 scleroderma and 19 polymyositis. IgA anti-afodrin antibodies were tested by ELISA.Results: Anti-afodrin antibodies were detected in 29 patients (5.9%): 6/209 primary SS (2,7%) and 23/282 secondary SS (8,2%), 13/190 RA (6.8%), 6/60 SLE (10%) and 2/19 Polymyositis (10,5%). No significant differences were observed when we compared patients with and without anti-afodrin antibodies, neither in epidemiological data, in time of disease evolution, nor in clinical manifestations.Conclusions: In our environment, IgA anti-alpha-fodrin antibodies are not common in patients with SS (5.9% vs 90-60% of the literature), and are not associated with any specific clinical manifestation. Sensitivity of IgA anti-alpha-fodrin antibodies is higher in secondary SS. (RA) is a systemic autoimmune disease characterized by chronic synovial joint inflammation leading to cartilage and bone destruction. The pathogenic role of TNF can be evidenced by: It has been observed that in plasma, synovial fluid and tissue of patients with RA high concentrations of TNF are found; transgenic mice that over-express the human TNF gene develop a polyarthritis similar to RA, while the administration of human TNF monoclonal antibodies from their birth, prevent the articular lesions and diminish the incidence of murine arthritis and, probably the most notable piece of evidence, comes from studies in which it has been demonstrated the clinical benefit of patients with RA treated with anti-TNF monoclonal antibodies or with soluble receptors of TNF. OBJECTIVE: The purpose of this work is to study the historic evolution of RA, which it would be considered an increasing disease, relatively new, favored by a specific mutation at position-308 in the promoter region of the TNF gene. METHODS: 308 single nucleotide polymorphism (SNP) was determined by polimerase-chain reaction (PCR)-restriction fragment length polymorphism. RESULTS: The-308 SNP determines a higher expression of this cytokine. The frequence of this polymorphism is 43.5% in Caucasic population. In studies of association of de SNP-308 and RA, the positive findings have been: in Caucasic population the allele TNF2 is 3 times higher in patients with RA, than in healthy controls; a relation between the SNP-308 and the presence of extra-articular manifestations with rheumatic nodules; in Swedish patients it has been demonstrated that individuals bearing the heterozygous form, develop a more severe disease and at an earlier age, and a significant association with bad prognosis was found in Turkish patients with RA. Objective: Low mannan binding lectin (MBL) and C4AQ0 has been associated with systemic lupus erythematosus (SLE). We asked whether low MBL might predispose to SLE in members of multicase SLE families, where there is an overall increased frequency of C4AQ0. Methods: Low MBL was detected by measuring serum levels (ELISA) and genotyping for mutant structural (O) and promoter (LX) alleles (RT-PCR). C4AQ0 was detected by protein electrophoresis. Twenty-four SLE patients from nine Icelandic families were compared to 83 first-degree and 23 second-degree non-SLE relatives, and 24 unrelated family members served as controls. Results: MBL-low (wild-type/O) and MBL-deficiency (O/O, LX/O) genotypes were associated with MBL levels below 1000 Ag/L, and low MBL was observed in five of the nine families (n = 86). In accordance with MBL genotypes, patients from these families also had lower MBL levels than their relatives (P b 0.001) and controls (P = 0.02). There was no evidence of MBL consumption. Conclusion: MBL-low/deficiency genotypes and low MBL serum levels predispose to SLE independently of C4AQ0. Low MBL was absent in four of the nine families, highlighting the heterogeneity of SLE. Abnormal Dendritic Cell Activation in SLE. H. Zhuang, 1 D. C. Nacionales, 1 S. Narain, 1,2 E. Sobel, 1,2 P. Y. Lee, 1 Circulating peripheral blood mononuclear cells (PBMCs) from SLE patients express high levels of Type I interferon (IFN-I) inducible genes. IFN-I is produced by plasmacytoid dendritic cells (PDCs) and promotes the maturation of myeloid dendritic cells (MDCs), which play a key role in antigen presentation. We investigated the interrelationships between IFN-I production and circulating PDCs and MDCs in subjects with SLE (n = 88), other autoimmune diseases (n = 82), and healthy controls (n = 57). Expression of the IFN-inducible genes Mx1 and OAS (real-time PCR) was increased in SLE PBMCs vs. the other groups (P b 0.0001, ANOVA). In contrast, circulating PDC and MDC counts (flow cytometry) were decreased in~50% of SLE patients compared with controls (P b 0.0001, ANOVA). Production of autoantibodies against dsDNA and snRNPs (Sm/nRNP, Ro, and La) was positively associated with high IFN-I production and negatively with the numbers of circulating PDCs and MDCs, whereas antiphospholipid antibody production was negatively associated with IFN-I production. Patients with low PDC/MDC counts fulfilled a greater number of ACR criteria and had a higher prevalence of renal disease. To better understand the basis for the low numbers of circulating dendritic cell precursors in SLE, we asked whether the PDC/MDC counts were persistently or intermittently low. These differences were independent of respiratory infections, SLEDAI, and medication use. However, PDC and MDC counts tended to remain low in SLE regardless of the IFN-I levels. Healthy subjects exhibited a different pattern: IFN-I expression was generally much lower at baseline, increased dramatically within 1-2 days in response to viral upper respiratory infections, and returned to baseline within 10 days; interestingly, the numbers of circulating PDCs/MDCs did not decrease during viral infections. Studies in a mouse model suggested that the low circulating PDC/MDC counts in lupus are due to enhanced dendritic cell maturation (increased CD86 expression) and migration from the circulating pool to sites of inflammation. The abnormal dendritic activation in SLE could reflect either an intrinsic dendritic cell defect or an exaggerated response to extrinsic (microbial?) Marginal zone (MALT) lymphomas arising in the stomach sometimes bear immunoglobulin receptors specific for Helicobacter pylori antigens and eradication of the infection may lead to tumor regression. Marginal zone lymphomas also arise in the salivary glands of patients with SjogrenTs syndrome (SS). We studied two SS patients with B cell neoplasms consistent with marginal zone lymphomas originating within the parotid gland. Both patients were positive for anti-Ro52 autoantibodies using a recombinant human Ro52 based ELISA. The first patientTs lymphoma exhibited typical lymphoepithelial lesions on H&E staining and was k L-chain + , CD5-, CD10-, and CD23-by flow cytometry. The solitary tumor was excised surgically after which her anti-Ro52 IgG autoantibody level fell from 735 units to 414 units. In contrast to the total IgG, which exhibited a k/l ratio of 1.5:1, the k/l ratio of her anti-Ro52 antibodies was~60:1. The k L-chain + anti-Ro52 antibody level decreased by more than 40% after surgery whereas the l Lchain + anti-Ro52 remained unchanged indicating that excision of the tumor specifically decreased the k L-chain + anti-Ro52 antibodies. The second patientTs lymphoma was l L-chain + with surface markers compatible with a marginal zone lymphoma. The neoplasm was too widespread at the time of diagnosis to permit local excision. The ratio of k/l L-chains in total immunoglobulin from Patient 2 was 8:1, whereas the k/l ratio of her anti-Ro52 autoantibodies was 0.5:1. Thus, both the tumor and most of her anti-Ro52 antibodies were l L-chain + . These data suggest that the production of anti-Ro52 antibodies was dependent on the presence of tumor cells (Patient 1) and that the anti-Ro52 antibodies exhibited non-random usage of L-chains (Patients 1 and 2), consistent with the possibility that the neoplastic B cells produced autoantibodies against Ro52. By analogy with marginal zone lymphomas arising in the stomach that are specific for H. pylori antigens, we speculate that some marginal zone lymphomas originating in the salivary glands may be stimulated by the self-antigen Ro52. These cases raise the possibility that anti-Ro52 autoantibodies, a specificity characteristic of SjogrenTs syndrome, can be produced by marginal zone B cells and that these cells may on occasion undergo neoplastic transformation. Background: Interleukin-1 (IL-1) is a prototype of a proinflammatory cytokine in that it induces expression of a variety of genes and synthesis of several proteins that, in turn, induce acute and chronic inflammatory changes. Polymyositis and dermatomyositis are chronic inflammatory muscle disorders, characterized by proximal muscle weakness and by inflammatory cell infiltrates in skeletal muscle. Increased expression of IL-1alpha in endothelial cells of capillaries and IL-1beta in mononuclear inflammatory cells, is a consistent finding in muscle tissue from myositis patients. The pathophysiologic role of these cytokines in myositis has not yet been clarified. Our hypothesis is that IL-1 could have a negative effect on muscle fibre metabolism and regeneration and contribute to the persisting muscle weakness and muscle fatigue often seen in these patients.Aim: To investigate if muscle fibres express IL-1 receptors (IL-1R) in healthy or inflamed muscle tissue and if so, if there was any difference of IL-1 receptor expression between symptomatic and non-symptomatic muscle tissue.Method: Muscle biopsies from 8 polymyositis patients, 3 dermatomyositis patients, and 6 healthy controls were included in this study. The muscle biopsies from the patients were taken from two different sites, one from a symptomatic muscle and the other from a non-symptomatic muscle. IL-1alpha, IL-1RI, and IL-1RII expression was investigated by immunohistochemistry. Localization of IL-1RI and IL-1RII expression was also investigated by double staining and confocal microscopy with laminin to identify muscle fibre membrane and a the marker BOBO3 to identify nuclei.Results: IL-1RI and IL-1RII were expressed, in the membrane and in the nuclei of muscle fibres as well as in inflammatory cells and endothelial cells in muscle biopsies from myositis patients. Healthy controls only had a scattered pattern of IL-1RI and IL-1RII expression in a few endothelial cells and in a few nuclei of the muscle fibres. There was no difference between symptomatic and asymptomatic muscles. IL-1a was expressed in endothelial cells and inflammatory cells in the patients.Conclusion: This is the first time that IL-1RI and IL-1RII have been described in the membrane of human muscle fibers. Furthermore, IL-1Rs were localized to cell nuclei. The implication of this is not known, but IL-1alpha is known to have an intracrine pathway and this could possibly be mediated through nuclear receptors. The observed expression of IL-1RI and IL-1RII in muscle fiber membranes supports our hypothesis that IL-1 could have a direct effect on muscle fibres and thereby affect muscle function. TRAIL is a member of the TNF family with proapototic activity. Increased T cell associated and soluble TRAIL (sTRAIL) levels in serum from patients with systemic lupus erythematosus (SLE) have been previously reported. In this study we set to characterize the upregulation of both T cell associated and sTRAIL in vivo and the modulation of TRAIL expression and soluble protein release following T cell activation and IFNa exposure in vitro. Using flow cytometry the ex vivo expression of membrane bound TRAIL was higher on CD4+ and CD8+ T cells from ten lupus patients compared to ten healthy controls, particularly on activated CD69+CD8+ T cells. sTRAIL levels determined by ELISA in sera from 34 lupus patients and 26 controls were significantly elevated in patients with active SLE and correlated with disease activity and with levels of IFN a but not with any particular clinical feature. In vitro, both T cell associated and sTRAIL were maximally induced by T cell activation plus IFN a in patients and controls. Western blot analysis of immunoprecipitated sera demonstrated the presence of the 21 kD monomeric forms but not of multimeric forms of sTRAIL. Although both Tcell associated and sTRAIL were functional in vitro in inducing apoptosis of TRAIL sensitive Jurkat cells as determined by Annexin V staining and 51Cr release assay, the apoptotic activity of membrane TRAIL was 2.5 fold higher compared to that of sTRAIL. In conclusion, increased T cell associated and sTRAIL is a feature of active disease in lupus patients and likely reflects the in vivo T cell activation and IFN a production. Collagen-PVP subcutaneous administration to RA patients was safe and well-tolerated drug for the short termtreatment. Sa1 AIM: To determine the efficacy, tolerance and safety of intramuscular injections of porcine type I collagen-PVP in patients with RA in a long term-therapy.METHODS: The protocol was approved by the Committee of Medical Ethics of the National Institute of Medical Sciences and Nutrition. Only patients who gave informed consent to participate were recruited. The study was double blind placebo-controlled and included 30 patients who fulfilled the 1987 American Rheumatism Association (ACR) criteria for active RA. Patients on stable therapy with methotrexate and/or non-steroidal antiinflammatory drugs (NSAIDs) were enrolled in a 1 year prospective, comparative and longitudinal study. Patients were treated in accordance to Freyberg scheme with intramuscular injections of 2 ml of collagen-PVP (3.4 mg of collagen) or 2 ml of placebo during 6 months. The primary endpoints were done according to Ritchie index (RI, 72joint count), 72-swollen joint count, disease activity score (DAS), erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP). The improvement was determined using American College of Rheumatology response criteria (ACR20, 50 and 70). Statistical analysis was performed by the non-parametric double tailed Mann-Whitney U test. Data were expressed as the mean F SD. The p values smaller than or equal to 0.05 were considered as significant.RESULTS: Collagen-PVP was safe and well tolerated. There were no adverse events. Patients had a statistically significant improvement (P b 0.05) in collagen-PVP-treated vs. placebo at 6 months of treatment in: swollen joint count (8.2 F 0.8 vs. 14.9 F 1.6; D-14, À55% vs. D-10, À41%), RI (10.6 F 0.8 vs. 15.2 F 1.5; D-19.1, À60% vs. D-13.6, À45%), morning stiffness (9.6 CONCLUSION: Collagen-PVP has been shown to be a safe and well-tolerated drug for the long-term treatment of RA. That the National Reference Laboratory (NRL)-Philippines shall establish partnership with National Epidemic Sentinel Surveillance System (NESSS)-National Epidemiology Center-Department of Health, Philippines.Materials and Methods: A network of hospital sentinel are to the Regional Epidemiology and Surveillance Units. The hospital have a laboratory capacity to do malarial smears, blood and stool cultures and hepatitis serology, participating authorities, surveillance personnel and availability of communication means between sentinel sites and regional epidemiology and surveillance units.Hospital admission are the basis in monitoring occurences of diseases, thus, a rapid, timely, accurate information and early warning on the disease outbreak.Quality Assurance procedures are ensured in each sample collection and during transport to NRLs. laboratory management includes Quality Control sampling to monitor accuracy and precision of existing procedures.Results: Sixteen (16) RESU were involved nationwide and all the Five (5) designated NRLs-Philippines conducted the screening and specific/confirmatory tests.All data from these surveillance and quality evaluation were extensively used utilized by the Department of Health for policy formulation and program evaluation, thus were able to contain the infectious disease identified.CONCLUSION: A quality assurance survey which is essential in the containment of infectious disease with outbreak potential has been established by the National Reference Laboratory-Philippines. This system will enable monitoring of precise and accurate screening and confirmatory tests of the disease and therefore a ready detection and containment if not eradication. Background: Tuberculosis is one of the most common infectious diseases in the world. One of the interesting gene for investigator is IFN-gR1. Clinical Characteristics of 88 Patients with Juvenile or Adult Dermatomyositis. Introduction Dermatomyositis (DM), belonging to the group of the idiopathic inflammatory myopathies, is characterized by a bimodal pattern of age-specific incidence of rates, with peaks in the age group from 5 to 16 years (juvenile DM) and in the age group from 45 to 65 years (adult DM). The aim of this study is to evaluate the clinical characteristics of 22 patients with juvenile DM.Methods A national registry of patients with juvenile dermatomyositis (JDM) was elaborated by the authors in Hungary. We summarize data of the register according signs and symptoms, disease course, frequency of relapses and survival of patients with JDM. Analysis was performed using data for 22 patients diagnosed between 1976 and 2003 according to Bohan and PeterTs criteria. Survival probability was calculated by Kaplan-Meier method. Data of juvenile patients were compared with data of 66 patients with adult DM.Results All children had symmetrical weakness of the proximal muscles. The most frequent cutaneous features were facial erythema and GottronTs papules (19/22). Extramuscular and extraskeletal manifestations of the disease were more frequent in adult patients. Only one patient with juvenile DM had interstitial lung disease (ILD). Cardiac manifestation of the disease or respiratory muscle involvement was not observed in juvenile patients. Respiratory muscle involvement (12/66) and ILD (11/39) were more frequent among adult DM patients than the cardiac manifestation of the myositis (6/55). In view of the disease course, the authors found that frequency of polycyclic and monophasic subtypes of the disease were similar. The hazard of the relapse was found higher during the first year after the remission. Among adult patients 4 disease-specific deaths occurred.Conclusion Patients with JDM are usually admitted to Dermatology departments, therefore paediatric dermatologists should be familiar with the clinical presentation of JDM. We report the first study on clinical characteristics and disease course of patients with juvenile DM who were diagnosed, treated and followed-up in Hungary. Studies of early vertebrates have provided clues to the development of the adaptive immune system, and even more primitive animal phyla, the Porifera, express molecules that are precursors of mammalian innate immunity. To gain new insight into potential mechanisms of autoimmunity in humans, we have studied a cytokine-like molecule, pre-B cell colony enhancing factor (PBEF), a protein produced by sponges in response to xenogeneic cells and with nicotinamide (Nam) phosphoribosyl transferase activity in both bacteria and mammals. PBEF was induced in B lymphocytes by activation of surface immunoglobulin receptors and IL-4 and stimulated production of IL-8 and other proinflammatory mediators by monocytes through an NF-kB dependent pathway. Consistent with its homology to Nam phosphoribosyl transferase, PBEF induction of IL-8 was inhibited by Nam. To determine whether PBEF might be overexpressed in patients with autoimmune disease, PBEF mRNA was measured by quantitative real-time PCR in PBMC from 89 patients with systemic lupus erythematosus (SLE), 22 patients with rheumatoid arthritis (RA), and 28 healthy donors (HD). In summary, our data demonstrate that PBEF is a cytokine-like inducer of proinflammatory mediators that utilizes the nicotinamide adenine dinucleotide and NF-kB pathways to stimulate innate immune system activation. Increased production of PBEF in autoimmune diseases may represent a mechanism that promotes inflammation and tissue damage and may be a rational target for therapeutic inhibition. Objectives: 1) to compare the MBL2 genotypes in RA versus healthy controls, and 2) to investigate the associations with radiological progression rate and serological markers.Patients and methods: MBL2 genotyping (INNO-LiPA MBL2, Innogenetics, Ghent, Belgium) was performed in 166 RA patients and 172 healthy controls. All RA patients were tested for anti-cyclic citrullinated peptide antibodies (anti-CCP2 antibodies, Eurodiagnostica, Arnhem, The Netherlands), rheumatoid factor (RF, Latex Fixation assay, DifcoLaboratories, Detroit, MI) and shared epitope (INNO-LiPA HLA-DRB1 or-DRB decoder amplification kits, Innogenetics, Ghent, Belgium). The Mann-Whitney U test was used for comparing the different combined haplotypes. P-values b0.05 were considered significant.Results Conclusions: In our study, neither RA susceptibility nor radiological prognosis was associated with a specific MBL2 genotype. Furthermore, RA-associated serological markers were equally found in the different MBL2 genotypes. Many studies have indicated a role for the type I interferon system in both human and murine systemic lupus erythematosus (SLE). In the anti-CD1 T cell receptor transgenic lupus mouse model (CD1 SLE model), single positive (CD4+ or CD8+) T cells from transgenic BALB/c donors induce a lupus-like syndrome when transferred to irradiated BALB/c nu/nu recipients. Disease in these mice is characterized by the presence of anti-dsDNA antibodies, proteinuria, and immune complex glomerulonephritis. Immunoprecipitation studies have revealed that a prominent target of serum autoantibodies in CD1 SLE mice is an interferoninducible antigen that has been identified as a member of the interferon-inducible 200 (Ifi200) family. We therefore hypothesized that IFN-inducible proteins are also targeted by serum autoantibodies derived from human lupus patients. We screened a panel of monoclonal and polyclonal antibodies directed against well-characterized lupus autoantigens by Western blot and found that the expression of Ifi16 and Ro52 proteins is inducible in HT1080 cells upon treatment with IFN-a and that the expression of both proteins peaks at 24 hours following treatment. The expression of both Ifi16 and Ro52 is also induced following treatment with IFN-h, another type I IFN. HT1080 cells were treated with IFN-a for 24 hours, lysed, and probed by Western blot with sera derived from 15 SLE patients. Serum from a single patient uniquely targeted an unidentified 32 kD-interferon inducible protein. Finally, the expression of Stat1, an inducible protein involved in both type I and type II IFN signaling, was elevated in PBMCs derived from SLE patients as compared to normal controls. Taken together, the data suggest that IFNinducible proteins represent a novel class of autoantigens and reveal a potential link between the type I IFN system and autoimmunity. Nuclear receptors are recognized as regulators of inflammation. PPARg is a nuclear receptor that was previously considered an orphan receptor but is now studied for its role in many biological processes. PPARg agonists are inhibitors of inflammatory mediators including nitric oxide (NO), TNF-alpha, and interleukin 6. Because of these early reports, the thiazoledinediones (TZDs) and other synthetic PPARg agonists are being studied in both animal models and human patients for their effects on inflammatory diseases such as systemic lupus erythematosus (SLE) and multiple sclerosis. However, intrinsic PPARg function in the absence of synthetic agonists appears to play an endogenous role in inflammatory modulation. It is thought that PPARg competes with other nuclear factors, like NFkB for common cofactors and activator molecules by a mechanism called transrepression. Our interest is in determining if such transrepression occurs between nuclear hormone receptors, specifically estrogen receptors (ER) and PPARs in inflammatory conditions. Such competition between the estrogen receptors and PPARs may partially account for gender differences in inflammatory diseases. Our current studies demonstrate that like activation of PPARg, activation of PPARy with GW610742X at 10-20AM is effective at significantly reducing NO production by 50% in LPS stimulated RAW246.7 macrophages. Since RAW246.7 macrophages are reported not to express PPARg, this suggests that PPARy may be an effective regulator of inflammation. We elected to use a murine model of lupus for our studies due to 10 fold higher incidence of lupus in females compared to males. We bred the ERh -/-genotype onto the MRL/lpr lupus mouse background for 8 generations. We collected peritoneal macrophages and stimulated them with LPS to induce NO production. After treatment with the PPARy agonist GW610742X, we measured effects on NO production. GW610742X reduced NO production by macrophages at 10-20AM concentrations. NO reduction was most effective in macrophages from ERh -/-mice at 55% and least effective in ERh +/+ mice at 30%. Our results suggest that nuclear receptors like ERh may interfere with the anti-inflammatory properties exhibited by some of the PPARs. In addition these results demonstrate the anti-inflammatory properties of PPARy. Competitive nuclear receptors may be useful pharmacological targets for the treatment of inflammatory diseases like SLE. Heavy metals can induce autoimmune reactions by direct linkage with MHC molecules, by modifying membrane proteins, presentation of cryptopeptides, bcrushingQ autoantigen by toxic radicals of oxygene, increase activity of enzymes of nucleinic exchange. These statements have formed the basis for studying concentration of Fe, Cu, Zn, Co, Mn, Cr, Sr, Pb, Ba in blood serum of 116 patients with systemic sclerosis by inductive coupled plasma(ICP) method.It was established, that in patients with systemic sclerosis deficiency of Zn, Fe and Mn is observed, increased value of Cu, Co and Cr, indicates clear correlations between the desease duration, level of activity, character of clinical sympthomes and some immunological parameters. Copper concentration is mostly expressed (increase in 5 times in comparison with norm) and zinc (decrease in 3 times). Methods of treatment used in systemic sclerosis cases(in particular, penicylamine) promotes further zinc exhaustion on blood serum and cellular levels.The received results point to trace elements disbalance as pathogenetic basis of this desease. Gene expression profiling of active Systemic Lupus Erythematosus (SLE) mononuclear cells show a significant granulopoiesis signature that can be traced down to the presence of immature blood neutrophil precursors. Indeed, using antibodies against CD16 and CD11b, neutrophils can be separated into three stages: immature neutrophil (IN) stage I (CD16-/CD11b-); IN stage II (CD16-/CD11b+), and stage III or mature neutrophil (CD16+/ CD11b+). While in healthy individuals cells negative for either CD16 or CD11b are not found in the blood, blood neutrophils in SLE patients contain cells belonging to all three stages. We have successfully obtained RNA from highly pure populations of immature (stage I) and mature (stage III) SLE as well as mature healthy blood neutrophils. After RNA amplification, labeling and hybridization to Affymetrix U133 gene chip arrays, we have detected i) genes that are specifically transcribed in immature versus mature neutrophils, ii) neutrophil genes that contribute to the SLE-specific gene signatures. Furthermore, we have found that the presence of immature neutrophils correlates with SLE disease activity and with the development of renal disease, suggesting that they are relevant to disease pathogenesis and severity.The presence of immature neutrophils in the SLE blood could be a reactive process due to the apoptosis of mature cells. Indeed, we have found that the number of immature neutrophils in the SLE blood mononuclear fraction correlates with the ability of the patientsTs serum to induce apoptosis of healthy mature neutrophils. Whether anti-neutrophil antibodies and/or death-inducing molecules like TRAIL are responsible for the pro-apoptotic effect of SLE serum on healthy neutrophils is currently under investigation.We have also found that mature SLE neutrophils display accelerated spontaneous apoptosis when compared with healthy mature neutrophils in culture. Despite an increased apoptosis rate, mature SLE neutrophils promptly release as much IL-8 and MIP1alpha as healthy cells when stimulated with the TLR2 agonist lipopeptide and TLR7/8 agonist R848, implying that these cells are functional. The release of cytokines by pro-apoptotic SLE neutrophils could be contributing to an inflammatory environment which would facilitate the maturation of antigen-presenting cells and the processing of apoptotic material in an immunogenic manner, explaining some of the key pathogenic events in SLE. Objective: To determine whether oral contraceptive (OCP) use and cigarette smoking are associated with the presence of rheumatoid factor (RF) in a cohort of reproductive age women without rheumatoid arthritis (RA).Materials/Methods: Subjects were 297 women who were the parents of children enrolled in a cohort originally established for the prospective study of the development of type I diabetes mellitus-related autoimmunity. This parental population was selected because it is enriched with HLA-DR4, a susceptibility marker for both type I diabetes mellitus and RA. Subjects were interviewed and examined to rule out a current diagnosis of RA. Subjects who met 1987 American College of Rheumatology criteria for RA were excluded from the analyses. A questionnaire was administered to obtain data on current and past history of tobacco and OCP use. Serum samples obtained from the adults at the time of the interview were tested for RF by nephelometry. Logistic regression models were performed to determine the association between RF and pack years of smoking as well as OCP use. Adjusted odds ratios (OR) and 95% confidence intervals (CI) were used as the measures of association.Results: Subject age range was 23.1-53.7 years (mean: 38.1, median: 38.3). 89.6% of women reported ever using OCPTs, and the mean duration of OCP use was 7.3 years. Categories of smoking were Never (n = 204), 1-19 pack-years (n = 88), and z20 pack-years (n = 5 Conclusions: These data suggest that OCP use may protect against the development of RF in individuals without RA. Additionally, heavy cigarette smoking may be a risk factor for RF seropositivity in individuals without RA. Previous studies have proposed that smoking is a risk factor for RA development, and OCP use is inversely associated with RA development. Our results suggest that both of these environmental exposures may act very early in the development of clinically apparent disease.Sa1.105. Efficacy of Apratastat, a Novel Dual Inhibitor of TNF-A Converting Enzyme/Metalloproteinase, in Murine Collagen-Induced Arthritis Models. Orally bioavailable, small molecule inhibitors that block release of TNF-a present a highly desirable strategy for treating RA.Objectives: 1) To determine the effects of Apratastat, a novel dual TACE/metalloproteinase inhibitor in two CIA models. 2) To compare Apratastat in one CIA model with a broad-spectrum metalloproteinase inhibitor, having no significant TACE activity (1) .Methods: Collagen-induced arthritis (CIA) was induced in DBA/1 mice by immunizing at the base of the tail with bovine type II collagen (CII) emulsified in complete FreundTs adjuvant, and boosting with either lipopolysaccharide (LPS) or CII emulsified in incomplete FreundTs adjuvant 21 days later.Results: Apratastat was evaluated in CIA mouse models with either an LPS boost or a collagen boost. In a prophylactic regimen with an LPS boost, Apratastat was administered orally at doses of 5, 10, or 20 mg/kg BID from days 18 through 35 post inoculation. In a second CIA model with a collagen boost, therapeutic treatment was initiated in each mouse when the disease severity score was at least 1. Apratastat (10, 25, 50, or 100 mg/kg BID, and 200 mg/kg QD, PO), or MPI-369 (100 mg/kg BID, PO), a broad-spectrum metalloproteinase inhibitor having no significant TACE activity, were evaluated in the therapeutic regimen. Apratastat at 100 mg/kg BID or 200 mg/kg QD produced a significant reduction in clinical and microscopic disease severity scores. The lower doses of Apratastat were similar to the vehicle control.Conclusion: These data show that a potent TACE/metalloproteinase inhibitor, Apratastat, reduces the clinical and histological manifestations of joint destruction in 2 murine CIA models of immune-mediated arthritis. Systemic lupus erythematosus (SLE) is characterized by the production of autoantibodies and glomerulonephritis. The chronic GVH (cGVH) model of SLE is induced by allorecognition of foreign major histocompatibility complex (MHC) Class II determinants. Our studies using CD4KO mice showed that endogenous CD4 T cells are required for emergence of autoreactive B cells during cGVH. In new studies we have attempted to characterize which subsets of B cells are prone to losing tolerance in cGVH. B cell subsets were characterized phenotypically by expression of specific cell surface proteins. Thus, immature B cells were sorted as HSA hi IgM hi AA4.1 + IgD-while mature B cells were sorted as HSA lo IgM lo AA4.1-IgD + . These different B cell subsets were adoptively transferred into irradiated (300 rads) JHT recipients, which lack endogenous B cells because of a targeted deletion in their JH segment. cGVH was induced by challenging the recipients with bm12 spleen cells. Our data showed that mature B cells lose tolerance and produce anti-dsDNA autoantibodies sooner than immature B cells. To determine which subset of the transferred mature B cell population was susceptible to loss of peripheral tolerance during GVH, we utilized additional cell surface markers. IgM int IgD hi CD21 int CD23 hi CD1-CD9 lo were sorted as follicular (FO) recirculating B cells, while IgM hi Ig-D lo CD21 + CD23-CD1 hi CD9 hi were considered as MZ B cells, which are normally located at the junction of white and red pulps. In similar adoptive transfer experiments followed by induction of GVH, our data indicated that MZ B cells lost tolerance and secreted autoantibodies sooner than FO B cells. MZ B cells have recently been proposed to play a critical role in host defense against T-independent blood-borne pathogens and in spontaneous development of autoantibodies, and expanded MZ B cell populations have been characterized in several lupus-prone strains of mice. Our data now show that MZ B cells are particularly vulnerable to loss of tolerance in the cGVH model of SLE and thus, may be an attractive target for therapeutic interventions. Systemic lupus erythematosus (SLE) is a multi-systemic autoimmune disease characterized by a great diversity of clinical manifestations accompanied by a large number of autoantibodies. Twenty-seven patients with SLE were defined according to the American College of Rheumatology criteria and were monitored over 2 years for disease activity. The anti-dsDNA autoantibody was measured by Farr assay and the anti-oxLDL autoantibody was determined by a standardized EIA. Both the antibodies were measured twice a year over the time. The results showed that 18 subjects were positive (N 5 IU/mL), 8 subjects were negative and 1subject varied in the borderline for levels of anti-dsDNA autoantibody; and 11 subjects were positive (N 100 unit/mL), 10 subjects were negative and 6 subjects varied between positive and negative for levels of anti-oxLDL autoantibody in the all detection during the monitored period. Six out of 27 patients were allnegative levels of both the autoantibodies. The levels of both autoantibodies substantially decreased in one patient and markedly increased in 4 patients over the time. The levels of anti-oxLDL autoantibody were significantly higher (P b 0.001) in the anti-dsDNA autoantibody positive group (119.4 F 49.2 unit/mL) compared to the anti-dsDNA autoantibodies negative group (82.9 F 37.7 unit/mL). There was a very significant correlation between the levels of anti-dsDNA autoantibody and the levels of anti-oxLDL autoantibody in the anti-dsDNA antibody positive group (R = 0.5856; P b 0.0001). There was no any correlation between both the autoantibodies in the anti-dsDNA antibody negative group (R = 0.0619). This study further suggests that oxidative damage is involved in the pathogenesis of SLE. It could be used as an associating marker in the monitoring of disease activity in SLE. The mechanism of elevated the levels of anti-oxLDL autoantibodies in active SLE subjects should be further study. Objective: Pregnancy-derived microchimerism has been implicated in the pathogenesis of autoimmune diseases that resemble graft-versus-host disease (GVHD). Evidence for fetal microchimerism (FMc) has been found in the blood and organs of women with systemic sclerosis and SLE. The mouse model for GVHD in which parental lymphocytes induce SLE-like glomerulonephritis and autoantibodies suggests that maternal microchimerism (MMc) may also be involved in SLE. We found maternal cells in the tissues of patients with autoimmune diseases and controls in the form of T lymphocytes, renal tubular cells, hepatocytes, and cardiac myocytes. Thus, persistence of MMc implies normal immune tolerance to maternal antigens, and loss of tolerance could lead to inflammation directed at MMc within tissues. Alternatively, clonal proliferation of maternal T lymphocytes reactive to host antigens could lead to elevated MMc in the peripheral blood. This study investigated MMc levels as well as tolerance to maternal antigens in childhood onset SLE. Reference Methods: Pediatric subjects were studied to avoid the potentially confounding factor of FMc. We used Real-Time Quantitative PCR specific for non-shared maternal HLA alleles to quantify maternal DNA in peripheral blood from 38 subjects: 14 with SLE and 24 age-matched, healthy controls. Lymphocyte reactivity was evaluated by intracellular cytokine assay and flow cytometry after stimulation of the probandTs peripheral blood mononuclear cells with CD14+ macrophages from the mother or an unrelated donor (URD).Results: Maternal DNA was detected in 21% of SLE patients and 38% of controls (OR 0.45, P = 0.17). After stimulation by URD macrophages 14% of CD4+ T lymphocytes from a healthy child produced interferon-g, whereas only 2% responded to maternal; 25% produced IL-4 in response to URD and 2% to maternal. In contrast, tolerance to maternal cells was not observed in a pediatric SLE patient: 10% of CD4+ lymphocytes produced IFN-g in response to URD macrophages compared to 20% to maternal, and 16% produced IL-4 to URD compared to 31% to maternal.Conclusions: MMc was common in the healthy subjects tested. In contrast to previous studies in which FMc and MMc was increased in autoimmune disease, there was a trend toward decreased MMc in pediatric SLE patients. In one SLE patient increased reactivity to maternal antigen presenting cells was observed, suggesting that MMc could be cleared from the circulation by host T cells. These preliminary results suggest the hypothesis that immunological tolerance to MMc is intrinsic to normal biology but may be lost in chronic inflammatory disease, leading to tissue-specific inflammation. Additional studies are needed to investigate MMc in tissues for frequency, morphology and phenotype and to extend initial observations suggesting a loss of tolerance to MMc in SLE. We present a 16 year old male with a history of retroperitoneal and mediastinal fibrosis. His case was complicated by renal and ureteral obstruction, and superior and inferior venae cavae obstruction with significant collateral flow. At that time renal function tests were abnormal, with ultrasound showing right hydronephrosis and CT scan showing stenosis of the inferior vena cava (IVC) between the infrarenal portion of the IVC and the hepatic portion, with extensive collaterals circulation. Ureteral stents were placed, and pulse doses of solumedrol were administered but the obstruction persisted. Previous reports have shown that the drug tamoxifen promotes activation-independent shedding of L-selectin (CD62-L) from neutrophils and lymphocytes, and it has been used in adults to treat this disease. The tamoxifen did result in resolution of his ureteral obstruction and removal of the stents. His course was complicated by a large deep venous thrombosis of the lower extremity and thrombosis of the SVC for which he continues to take warfarin prophylactically. Our patient has not had evidence of recurrence in the second month since discontinuation of tamoxifen, but he continues to be hypoxemic and oxygen dependent with physical activity. Pulmonary function testing is indicative of severe restrictive disease. Although his absolute B cell count is low, the percentage from the total counts is normal. We will be treating him with intravenous immunoglobulin (IVIG) in order to replace the deficiency and prevent infection. This is the first report of CVI in retroperitoneal fibrosis. Dendritic cells (DCs) are antigen presenting cells that play an important role in the immunopathogenesis of rheumatoid arthritis. DCs genetically modified have shown to reduce the severity of arthritis in the murine model of collagen induced arthritis (CIA). In addition, DCs expressing differential levels of MCH class II and co-stimulator molecules have demonstrated to interfere with the induction of CIA by a deficient presentation of antigen to T cells. Aim: To assess the capacity of DCs pulsed with peptides derived from type II bovine collagen (CII) to modulate the antigen-specific immune response in mice with active arthritis. Methods: After five days of culture with GM-CSF, bone marrow-derived DCs were incubated with LPS and then pulsed for 4 h with CII. DBA1/ lacj mice with active arthritis were subcutaneously inoculated either with CII-pulsed DCs or DCs alone. Both clinical and histopathological parameters were followed up for 70 days after the first CII administration. Results: Previous the immunization procedure, DCs were phenotipically and functionally characterized by MHC class II and co-stimulator markers and by a phagocytosis assay, respectively. In both analyses DCs displayed a semi-mature pattern. We observed that the group of mice with CIA and immunized with DCs alone showed a higher score of clinical signs than the control group of CIA (P b 0.05). However, the group of CIA mice immunized with CII-pulsed DCs showed a significant lower clinical score than CIA controls (P b 0.005). Conclusion: The immunization with semi-mature DCs pulsed with type II bovine collagen interferes with the immunological course of murine arthritis in an antigen-specific manner. IL-12 signaling through STAT-dependent pathway is essential for induction of IFNg and differentiation of Th1 cells. Mice expressing HLA-DQ8 in the absence of endogenous class II molecules develop severe collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis. The development of arthritis in DQ8 mice is T cell dependent with a Th1 profile. To understand the role of IL-12 in collagen-induced arthritis, we have generated Abo.DQ8 transgenic mice lacking STAT4. Both STAT4À/À and STAT4+/+ mice could mount response to collagen in vitro. However incidence of CIA was significantly higher in DQ8.STAT4+/+ mice compared to DQ8.STAT4À/À mice. The development of arthritis was dependent on Th1 cytokines as both transgenic mice produced IL-18 and TNF-a. DQ8.STAT4À/À mice produced very low levels of IFNg suggesting IL-12 controls the production of IFNg through a STAT4-dependent pathway. STAT4-/-mice developed milder arthritis with delayed onset than that observed in DQ8.STAT4+/+ mice. CIA in the absence of IFNg and IL-12, suggests that these cytokines might regulate the severity of disease. Mice carrying non-susceptible HLA-DQ6 transgene produce lower amounts of Th1 cytokines and develop milder CIA with lower incidence compared to DQ8 mice. This suggests that while MHC is the major predisposing factor, cytokines produced in response to antigen presented by the MHC molecule may determine severity of disease. Systemic lupus erythematosus (SLE) is caused by autoantibodies (e.g. anti DNA), and immune complexes causing organ damage. NZB/NZW Fl female (BWF1) mice tolerized with an artificial peptide (pCONSENSUS, pCONS) based on anti-DNA IgG sequences containing MHC Class I and Class II T cell determinants, develop regulatory CD4+CD25+ T cells and inhibitory CD8+ T cells, both of which suppress autoAb production. One to one comparison of white blood cells (WBC), CD4, and CD8 cell subsets from tolerated vs. non-tolerized mice showed 448, 174 and 60 genes that are differentially expressed by at least two-fold, respectively. From the CD8+ T cell arrays, we confirmed upregulation of several genes using real-time PCR. Increased expression pattern (more than two fold) with real -time PCR of 6 genes in CD8+T cells from tolerized mice was repeatedly found (IFI202B, Trp-53, Foxp3, CCR7, bcl2, and IFNar1). Immunophenotyping and cell sorting revealed significant expansion of CD3+CD8+, after pCONS treatment in BWF1 mice. Significant increased mRNA expression of TGFb and Foxp3 was found in CD8+CD28+Ti stimulated with anti-CD3/CD28 after pCONS treatment, suggesting a role of TGFb and Foxp3 in the suppressor function of these CD8+ T cells. Anti-TGFb abrogated suppression of anti-DNA production in vitro by CD8+ T cells cocultured with syngeneic CD4+ T helpers and B cells. Intracellular staining revealed increased expression of IFN-g and Foxp3 in splenic CD8 T cells from tolerized mice compared to those from untreated mice. Furthermore, Annexin V and 7AAD staining showed significantly less apoptosis in tolerized CD8+Ti cells than in naive. CD8+ Ti from tolerized mice exposed to si RNA of Foxp3 lost their ability to suppress anti-DNA production in vitro. This confirms the role of Foxp3 in the suppression associated with this model of immune tolerance. The role of other upregulated molecules are being studied in a similar fashion. We conclude that the suppressive function of tolerized CD8+ T cells relates to increased survival and secretion of TGFb, and to upregulation of Foxp3. Other gene products upregulated in these cells may also relate to their ability to survive and suppress autoimmunity. These studies on the molecular mechanisms of suppression of lupus in BWF1 mice may have potential therapeutic value in human SLE.Sa1.113. Impact of Gender on Immune Nephritis. Background: We have recently reported that the co-administration of anti-glomerular sera with LPS leads to severe nephritis, as gauged by proteinuria, azotemia, glomerular crescent formation, and proliferative lesions. The focus of this work is to ascertain how gender might impact nephritis in this model. Methods: Male and female C57BL/6 mice (some of which were deficient in estrogen receptors) were challenged with anti-glomerular serum plus LPS, and monitored for evidence of renal disease. In addition, some mice were subcutaneously implanted with 40-day release estradiol pellets (0.5mg), and then subjected to the nephrotoxic insult.Results: LPS and anti-glomerular antibody challenged female C57BL/6 mice exhibited significantly worse proteinuria (10.9 vs. 4.9 mg/24h at peak of disease, P b 0.007) and blood urea nitrogen (120.3 vs. 55.8 mg/dL at peak of disease, P b 0.029, N = 5 mice per group), compared to age-matched C57BL/6 male mice. Moreover estradiol-treated C57BL/6 males exhibited significantly worse proteinuria as early as D8 (16.7 vs. 11.8 mg/24h, P b 0.0005, N = 5-6 mice per group), compared to placebo-treated C57BL/6 males. Importantly, estradiol-treated C57BL/6 males that were deficient in ESR1 estrogen receptors exhibited significantly lower proteinuria (5.7 mg/24h), compared to estradiol treated C57BL/6 males (13.3 mg/24h, P b 0.004, N = 3-12 per group).Conclusion: It is clear that gender can profoundly influence immune-mediated nephritis, through the action of estrogens. Studies are in progress to determine the cellular and molecular bases for the differences. The pathogeny of primary SjfgrenTs syndrome (pSS) is still poorly understood. No comprehensive, systematic study of genes expressed or repressed in salivary gland, one of the main target-organ of patients with pSS, has ever been reported. We aimed to compare the gene-expression profiling in salivary glands of patients with pSS with that of subjects without any auto-immune features.Patients and Methods. Patients with pSS according to European-American consensus group criteria were included in the study, as well as patients withSS and rheumatoid arthritis and subjects without any feature of autoimmunity (no autoantibody, focus score b 1). Total RNA was isolated from labial salivary glands and RNA levels, quality and purity were assessed with the use of the RNA 6000 Nano assay on the Agilent 2100 Bioanalyzer. RNA was amplified (one-round amplification, Ampliamp, Ambion, UK), controlled on the BioAnalyzer, and c-DNA fluorescent probes were prepared from 250 ng of amplified RNA. CDNA fluorescent probes were hybridized to custom chips, containing 11000 cDNA spots (manufactured by the Commissariat à lTEnergie Atomique, CEA, France). RNA from a pool of 4 normal parotid samples was amplified and used as a common reference. All intensity values were normalized using LOWESS. P values were corrected for multiple comparisons according to Benjamini-Hochberg False Discovery Rate procedure.Results. Thirty-two microarrays were hybridized, using salivary glands of 7 patients with pSS, 7 subjects without any feature of autoimmunity and 2 patients with SS and rheumatoid arthritis. Taking into account features present in all microarrays, 169 genes (93 genes up-regulated, 76 genes downregulated) were differentially expressed between patients with pSS and subjects without any feature of autoimmunity. 612 genes were differentially expressed between these 2 groups after algorithmic evaluation of missing values. Hierarchical clustering showed that 6/7 patients with pSS clustered tightly together, as well as 6/7 subjects without any feature of autoimmunity. The 169 genes mainly belonged to the cell-cycle, oxidative burst, apoptosis, protein metabolism and chemokine families. Validation of these results using quantitative RT-PCR and immunohistochemistry are currently under way.Conclusion. This gene-expression profiling study in the target organ of pSS, which fulfilled the best standards for microarray data, demonstrates that the transcriptomic approach could be very helpful to unravel new pathogenic pathways in autoimmunity. Four Systemic Lupus Erythematosus Autoantigens Identified by HEp-2 Library Screening. Objective: To determine and characterize autoantigens that stimulate autoantibody production in systemic lupus erythematosus (SLE) by screening human epithelioma-2 (HEp-2) expression library with patient sera.Materials and Methods: Forty pediatric SLE patients with nephritis were screened for high titer anti-nuclear antibodies (ANA) by immunofluorescence and Western blot using HEp-2, leukemia (acute lymphocytic and myelocytic leukemia), and normal cells. Two sera yielded extremely high-titer ANA. Column-purified immunoglobulin G (IgG) samples from these two pediatric patients (both with diffuse proliferative glomerulonephritis) were used to screen a HEp-2 cell line expression library made with a Zap II cDNA synthesis kit by Stratagene. After immunoscreening, phagemids from the lambda ZAP-II vector were excised, amplified by PCR, characterized by agarose gel electrophoresis, and sequenced. Sequenced samples were identified by the National Center for Biotechnology Information (NCBI) BLAST program.Results: Out of the seventeen clones that were isolated, seven were identified by both sera. After screening out vector and repeat sequences, four unique antigens were identified: melanoma antigen gene Xp-2 (MAGE Xp-2), ribosomal protein P0, ribosomal protein P1, and ribosomal protein S6.Conclusion: Four autoantigens in systemic lupus erythematosus were identified by HEp-2 expression library screen. Screening different tissue expression libraries with patient sera may further characterize SLE autoantibody antigen specificities, improve our understanding of the pathogenesis of autoantibody production and end-organ damage in SLE, and may lead to the development of novel therapeutic interventions.Objective: External application of the extract of two herbs from two genera, Axonopus and Ludwigia, abbreviated as A/L extract, has been used to treat patients with second to third degree of burn injuries with high efficacy for decades. The main symptoms after severe burn are redness, pain, blister, and swelling due to inflammation; therefore, antiinflammatory medication is a basic and critical treatment. Hence, the aim of our studies is to determine whether A/L extract has anti-inflammatory activities.Methods: Non-adherent peripheral blood leukocytes (NA-PBL) and monocytes were isolated from human whole blood collected from three healthy donors. Human NA-PBL and monocytes were then stimulated with PHA with or without the presence of filtersterilized A/L extract. Supernatant was collected at 48hr and 72 hr for the measurement of TNF-alpha and IL-2 by ELISA. The cells were also stained with MTT at 72hr for proliferation assay.Three groups of mice after 2 nd or 3 rd degree of burn by boiled water were treated with placebo, 0.1% A/L extract, or 0.5% A/L extract daily.Results: In vitro studies from human NA-PBL and monocytes stimulated with PHA show that the incubation of the cells with A/L extract reduced the production of IL-2 (30% for NA-PBL, 48% for monocytes at 48 hrs) and TNF-alpha (~15% for both NA-PBL and monocytes at 48 hours and 35% for NA-PBL and 20% for monocytes at 72 hours). The proliferation assay shows that the cell prolifereation stimulated by PHA was inhibited by 25% with the presence of A/L extract. The results from the mice with burn injuries show a dose response: it took the mice (n = 7) treated with placebo about a month to heal, 3 weeks for the group (n = 6) treated with 0.1% A/L extract and 15 days for the group (n = 7) with 0.5% A/L extract. In independent experiments, the treatment of A/L extract on the mice with severe inflammation on whole back and whole limb also reduced the symptoms of inflammation, such as redness and swelling, within an hour; so did the results from the volunteers with pain on joint.Conclusion: The results from both in vitro and in vivo studies suggest that external application of A/L extract is able to reduce both superficial and sub-dermal inflammation. The external application to treat sub-dermal inflammation, such as Rheumatoid arthritis, will greatly reduce any risk of side effects. Further efforts will be made to study the effect of A/L extract on synoviocytes and the safety of oral usage to treat systemic inflammation. Identification of the disease-regulating T cell determinants within an antigen targeted in an autoimmune disease would be of significance both in better understanding of the mechanism of natural remission/recovery from acute phase of the disease and in developing antigen-specific immunotherapeutic approaches for that disease. Although pathogenic epitopes have been identified in several self and foreign antigens, there is barely any information regarding bona fide regulatory T cell epitopes. Using the Lewis rat adjuvant-induced arthritis (AA) model of human rheumatoid arthritis (RA), we have defined disease-regulating T cell epitopes within the AA-related antigen, mycobacterial heat-shock protein 65 (Bhsp65), and in its self homolog, rat hsp65 (Rhsp65). These epitopes reside within the C-terminal region of hsp65. Interestingly, despite sequence differences between the corresponding homologous C-terminal epitopes of the foreign (mycobacterial) and self hsp65, these epitopes are not only immunogenic and crossreactive with each other, but they also possess comparable AA-regulating properties. Intriguingly, the C-terminal epitopes of the self hsp65 are well processed and presented (dominant) from the native antigen, whereas those of the foreign hsp65 are poorly processed and presented (cryptic or hidden). However, like Bhsp65, Rhsp65 is also a good immunogen, showing that Lewis rats are not tolerant to either hsp65. Our results support a model for immunoregulation of autoimmune arthritis in which arthritic Lewis rats having inflammatory acute AA upregulate the expression as well as presentation of endogenous self hsp65 leading to priming of ambient C-terminal epitope-reactive T cells. These T cells then contribute to the downregulation of acute AA. Concurrently, the cryptic epitopes of Bhsp65 are efficiently processed under the inflammatory milieu of acute AA, and thereby, activate subsets of T cells directed against Cterminal epitopes of Bhsp65. These T cells are then activated and expanded by C-terminal epitopes of Rhsp65, and vice versa, resulting in a reinforcement of the regulatory T cell activity against AA. These results describe a novel diseaseregulating feature of cryptic antigenic determinants contrary to their well-known pathogenic attribute observed in other antigens. Thus, cryptic epitopes of autoimmune diseases-related antigens can be exploited for immunotherapeutic purposes in RA and other autoimmune diseases. Systemic lupus erythematosus (SLE) is a debilitating autoimmune disease that affects women nearly nine times as often as men. The weakly estrogenic organochlorine pesticide chlordecone can accelerate the onset and severity of immune complex glomerulonephritis in female (NZB Â NZW) F1 (NZB/WF1) mouse model. Moreover, chlordecone exposure caused the earlier onset of serum IgG anti-dsDNA and anti-chromatin in ovariectomized NZB/WF1 mice, similar to the effects caused by exogenous exposure to 17-beta estradiol. This murine model of SLE is characterized by the spontaneous development of germinal centers (GCs), which play a key role in the maturation of the humoral immune response. Chemokine receptors CXCR4 and CXCR5 are important for GC dark and light zone organization, and cell adhesion molecules ICAM and VCAM-1 may play an important role in reducing GC B cell apoptosis. We therefore examined the effects of chlordecone and 17-beta estradiol on germinal center B cells using two-month old ovariectomized NZB/WF1 mice. Mice were subcutaneously implanted with sustained-release pellets containing 17-beta estradiol (0.025mg/ kg/day) or chlordecone (0.5 and 2.5mg/kg/day). Mice were sacrificed six weeks after implantation, and spleen cells were studied by flow cytometric analysis. Splenic B cells were also purified for proliferation assays using 3 H-thymidine and apoptosis tests with annexin-V and 7-AAD. We also found that both chlordecone and estradiol increased the percentage of B cells expressing the GC marker GL7 (P b 0.01 by ANOVA), the B cell co-stimulatory molecule B7-2 (P b 0.01), and the activation marker CD44 (P b 0.01 by estradiol and P b 0.05 by chlordecone). The expression of CXCR4 (P b 0.01), CXCR5 (P b 0.01 by estradiol and P b 0.05 by chlordecone), ICAM (P b 0.01), and VCAM-1 (P b 0.01 by estradiol and P b 0.05 by chlordecone) on splenic B cells was upregulated as well. On the other hand, neither chlordecone nor estradiol produced a significant increase in B cell proliferation, but they both significantly reduced splenic B cell apoptosis. These findings suggest that both chlordecone and estradiol may enhance autoimmunity through effects on the germinal center response. The spondyloarthropathies (SpA) are a heterogeneous group of diseases characterized by axial as well as peripheral enthesitis and arthritis and less frequently by a range of extra-articular manifestations. The strong association between HLA-B27 and the SpA, particularly ankylosing spondylitis (AS) in the general population and in first degree relatives of affected individuals confers this allele a significant role in disease susceptibility. Yet, the fact that only approximately 2% of HLA-B27 individuals develop AS and that a small proportion of patients with AS does not carry the B27 allele suggests other genes participate in the disease susceptibility. Of special interest is the IL-10 gene that codified a potent immune modulator. Thus, the aim of the present study was to investigate the allele distribution of IL10 promoter polymorphisms in SpA Mexican patients. One hundred and twenty-six patients with SpA (55 with undifferentiated SpA (U-SpA), 55 with AS, and 16 with reactive arthritis (ReA)) and 91 healthy controls were studied. The IL-10 promoter polymorphisms (positions-592, À819 and-1082) were determined by polymerase chain reaction-restriction fragment length polymorphism. Statistical methods included the Mantel-Haenzel, X 2 , FisherTs exact test, and Woolf method for odds ratio (OR). The allele and genotype frequencies of the three polymorphic sites were similar in the whole group of SpA patients and healthy controls. When clinical subgroups (U-SpA, AS and ReA) were compared to healthy controls, the results did not reveal significant differences in allele or genotype frequencies. These data suggest that IL10 promoter polymorphisms are not involved in the genetic susceptibility to SpA in Mexican patients. Congenital heart block is a passively transferred autoimmune disease where Ro52 antibodies are transported from the mother to the fetus in Ro/SSA positive women. The diagnosis of the mother may be SLE or SjfgrenTs syndrome. It has been shown that the Ro52 antibodies are necessary for disease development, and immunization of rat mothers with Ro52 derived peptides results in heart block in the pups. However, the fact that mothers of affected children can later give birth to healthy children despite persisting Ro52 autoantibodies indicates that another factor such as genes are involved in the penetrance of the disease. The aim of this study was to investigate this by ascertaining the MHC and non-MHC genetic influence in heart block development in an immunization-induced rat model of congenital heart block.Four rat strains, 3 sharing the same MHC region (RT1) haplotype (DA, PVG.AV1, LEW.AV1), and 2 with similar background genes but different MHC genes (LEW.L and LEW.AV1) were studied. Female rats were immunized with Ro52 or control protein and mated. The resulting pups (208 from Ro52 immunized mothers, and 105 from the control immunized mothers) were analysed for congenital heart block with extremitylead electrocardiography of conscious pups. Serum samples were taken throughout the study from both mothers and pups and epitope mapping of the antibody specificities using recombinant Ro52 and a panel of overlapping and mutated Ro52 peptides was performed.Analysis of the ECGs of the 313 born pups from the four strains showed that in the 3 strains sharing the same MHC region haplotype (DA, PVG.AV1, LEW.AV1) AV block I developed in 45%, 35% and 44% of the pups, respectively. In the Lew.L strain only 14% of pups were affected by AV block I. There was no significant difference in generated Ro52 antibody epitope specificity between the rat strains. The hearts of all 44 rat mothers and 24 of the pup hearts were collected for immunohistochemical investigation of inflammatory markers.Our study is the first to explore genetic influence in development of congenital heart block in animal models, and results show that genes found in the rat MHC region are of significant importance for the penetrance of congenital heart block, while non-MHC genes appear to have little or no influence on the disease. Identification of Disease Profiles for Rheumatoid Arthritis Using Antibody and Antigen Arrays.Autoimmune maladies, such as, rheumatoid arthritis are difficult to diagnose and most likely represent a collection of pathologies that give rise to the symptoms of each disease. Furthermore, there is no definitive set of markers for these diseases that can be monitored in real time to predict or assess the effect of any treatment. Simultaneous and serial measurements of hundreds of proteins in the blood and synovial fluid will be needed to make a differential diagnosis and to discover novel patterns that would stratify the diseases. Protein microarrays are well suited for this type of proteomic approach despite the fact that they are limited by the specificity of the affinity reagents (i.e., antibodies), and the measurement accuracy is dependent on the array performance, sample preparation, and assay reproducibility. We demonstrate an improved method for fabrication of antibody and antigen microarrays using a thermal inkjet printer and functionalized glass slides, and have addressed quality issues such as checking purity and integrity of proteins prior to deposition, printed array quality, cross-reactivity of the affinity reagents, and need for removal of the high abundant proteins. Using our optimized array platform we developed antibody arrays for detection of over 60 markers of inflammation, including autoantibodies, cytokines, chemokines, soluble receptors and enzymes. Forty samples of synovial fluid and serum from RA patients and controls were run on the arrays. The arrays demonstrated increased levels of known inflammatory markers in RA samples among them matrix metalloproteases and cytokines.Sa1.123. Autoimmune maladies, such as, rheumatoid arthritis are difficult to diagnose and most likely represent a collection of pathologies that give rise to the symptoms of each disease. Furthermore, there is no definitive set of markers for these diseases that can be monitored in real time to predict or assess the effect of any treatment. Simultaneous and serial measurements of hundreds of proteins in the blood and synovial fluid will be needed to make a differential diagnosis and to discover novel patterns that would stratify the diseases. Protein micoarrays are well suited for this type of proteomic approach despite the fact that they are limited by the specificity of the affinity reagents (i.e., antibodies), and the measurement accuracy is dependent on the array performance, sample preparation, and assay reproducibility. We demonstrate an improved method for fabrication of antibody and antigen micoarrays using a thermal inkjet printer and functionalized glass slides, and have addressed quality issues such as checking purity and integrity of proteins prior to deposition, printed array quality, cross-reativity of the affinity reagents, and need for removal of the high abundant proteins. Using our optimized array platform we developed antibody arrays for detection of over 60 markers of inflammation, including autoantibodies, cytokines, chemokines, soluble receptors and enzymes. Forty samples of synovial fluid and serum from RA patients and controls were run on the arrays. The arrays demonstrated increased levels of known inflammatory markers in RA samples among them matrix metallaproteases and cytokines. Background: Pediatric systemic lupus erythematosus (SLE) is a multisystem disease that significantly impacts quality of life (QOL) of children. SLETs impact on school attendance, an important outcome and potential predictor of other outcomes, has received little attention.Objective: Examine the relationship of school attendance with QOL, physical function, SLE activity and damage in children with SLE.Design/Methods: In this cross-sectional study, children with SLE (6-18 years) and parents completed child/parent versions of: Childhood Health Assessment Questionnaire (CHAQ), Pediatric QOL Inventory (PedsQL Generic 4.0 and Rheumatology 3.0 modules). Physician completed: SLE Disease Activity Index (SLEDAI) and Systemic Lupus International Collaboration Clinics/ACR Damage Index (SLICC). Number of days over the prior 30 the child missed school due to physical/mental health reasons was recorded using PedsQL family information form. Spearman correlations were determined between number of missed school days and other variables.Results: 24 children (23 girls) with SLE (mean age 15 F 3 years, mean education 9 th grade, mean SLE duration 46 F 30 months, SLEDAI 0-24, SLICC 0-6, median CHAQ 0.3, mean PedsQL-Generic 67 F 20), and 19 parents (median CHAQ 0, mean PedsQL-Generic 69 F 18) participated. 4 children were excluded because school attendance was inapplicable. Mean number of days too ill to play = 3 F 7 and mean number of days needed someone = 3 F 6 days. The number of missed school days moderately correlated with decreased QOL as reported by parents and as measured by the PedsQL-Generic module (r 0.56, p 0.02), but did not correlate significantly with Rheumatology module, CHAQ, SLEDAI, SLICC, or any of the child reported scores.Conclusions: Number of missed school days is correlated with decreased general QOL in children with SLE as perceived by their parents. However, parallel correlation between missed school days and overall QOL as perceived by children was not identified. Discrepant perception between parents and children warrant further investigation in a larger cohort. Lack of correlation of school attendance with other scales suggests that generic scale captures a less tangible element of SLE.Category Background: Pediatric systemic lupus erythematosus (SLE) is a chronic fluctuating disease significantly impaction quality of life (QOL). There is no pediatric SLE-specific health-related QOL tool. SLE heterogeneity, childrenTs evolving needs and expectations, and parent-proxy respondents complicate QOL measurement.Objective: Develop a brief, valid and easily understood SLEspecific pediatric QOL tool.Design/Methods: We developed Simple Measure of Impact of Lupus Erythematosus in Youngsters (SMILEYn) based on qualitative research on pediatric SLE-a 31 item tool, with parallel child/parent versions and 5-faces scale items. Children 2-18 years and parents completed child-parent versions of the SMILEYn, Pediatric Quality of Life Inventory (PedsQL) Generic 4.0 scale, and Childhood Health Assessment Questionnaire (CHAQ). Children also completed the Piers-Harris Self-Concept Scale. Correlations of child/parent SMILEYn versions with the corresponding versions of the above scales were determined by Spearman rank test.Results: 15 children (12 girls) with SLE (mean age 14 F 2 years, mean SLE duration 44 F 37 months, SLE disease activity index 0-20, mean self-concept 50 F 9) participated with mean SMILEYn score of 106 F 20. Significant correlations are indicated by asterisks (table) . Results and Conclusions: Numerical values obtained for the binding and the dissociation rate coefficients are linked to the degree of heterogeneity or roughness (fractal dimension, D f ) present on the biosensor chip surface. The binding and the dissociation rate coefficients are sensitive to the degree of heterogeneity on the surface. For example, for the binding of adipose tissue interstitial glucose, as the fractal dimension value increases by a factor of 3.31 from D f1 equal to 0.5720 to D f2 equal to 1.891, the binding rate coefficient increases by factor of 8.88 from k 1 equal to 0.0545 to k 2 equal to 0.4841. An increase in the degree of heterogeneity on the probe surface leads to an increase in the binding rate coefficient. A dual fractal analysis gives a better fit in most cases for the binding kinetics. Affinity (ratio of the binding to the dissociation rate coefficient) values are also presented.The values of binding rate coefficient, k, linked with the degree of heterogeneity existing on the biosensor surface provide a complete picture of the reaction kinetics occurring on the sensor chip surface. Type 1 diabetes mellitus is considered to be the typical autoimmune disorder resulting in the loss of pancreatic beta-cells with the consequent development of absolute insulin deficiency. However, the exact changes of the components of the immune system including impairments of different cytokines levels preceding the development of clinically overt type 1 diabetes mellitus remain not fully understood. Therefore, the aim of this study was to investigate the serum levels of interleukin-16 in patients with newly diagnosed type 1 diabetes mellitus. We studied 10 patients with clinically overt type 1 diabetes mellitus aged 8-15 years old before initiating of insulin therapy and 10 aged-matched healthy control subjects. Serum interleukin-16 levels were measured by specific immunoenzyme assay. We found that serum levels of interleukins-16 were significantly decreased in patients with type 1 diabetes mellitus compared to control subjects-94 F 9.99 pg/ml vs. 270 F 34.78 pg/ml (mean F SEM), respectively, P b 0.001. These data could explain the decrease of the number CD4+ T-lymphocytes in subjects with newly diagnosed type 1 diabetes mellitus which was revealed in our earlier studies as these lymphocytes represent the target cells for interleukin-16. We may hypothesize that revealed significant decrease of the interleukin-16 production could play a role in the pathogenesis of autoimmune insulitis and consequent clinical manifestation of type 1 gdiabetes mellitus. As organ-specific autoimmune diseases do not become manifest until well-advanced, interventive therapies must inhibit late-stage disease processes. Using a panel of immunogenic peptides from various g-cell antigens, we evaluated the factors influencing the efficacy of antigen-based therapies in diabetes-prone NOD mice with advanced disease. The ability of the major g-cell autoantigen target determinants (TDs) to prime Th2 responses declined by a 80% between 6 to 12 weeks of age, while the ability of immunogenic ignored determinants (IDs) of g-cell antigens to prime Th2 responses was unaffected by the disease process. The different patterns of TD and ID immunogenicity (even from the same g-cell antigen) may be due to the exhaustion of uncommitted TD, but not ID -reactive, T cell pools by recruitment into the autoimmune cascade. Therapeutic efficacy was associated with a peptideTs immunogenicity and ability to promote Th2 spreading late in the disease process, but not its affinity for I-Ag7 or its expression pattern (g-cell specific/nonspecific, or rare/abundant). Characterization of some IDs revealed them to be babsoluteQ cryptic determinants. Such determinants have little impact on T cell selection, leaving large precursor T cell pools available for priming by synthetic peptides. Traditional antigenbased therapeutics using whole autoantigens or their TDs cannot prime responses to such determinants. These findings suggest a new strategy for designing more efficacious antigen-based therapeutics for late-stage autoimmune diseases. Objectives: Non-alcoholic steatohepatitis (NASH) is a common chronic liver disease, leading to cirrhosis and hepatocellular carcinoma. It has been considered tumor necrosis factor a or adipocytokine is important in the pathogenesis of NASH, which has a feature of metabolic syndrome. Although NASH is characterized by necro-inflammatory changes, an essential role of the inflammatory cells has not yet been identified. To clarify the role of inflammatory reactions in the pathogenesis of NASH, we analyzed the composition of liver-infiltrating cells isolated from liver biopsy specimens of NASH. Methods: 26 patients with NASH and 23 with fatty liver (FL) were analyzed. We performed immunohistochemical staining using antibodies against CD3, CD4, CD8, CD56, CD68, and CD15. Oxidative stress was assessed by the expression of 4-hydroxy-2-nonenal (HNE). We counted the numbers of each population of liver-infiltrating cells (/mm 3 ). We diagnosed the liver histology using scoring system proposed by Brunt et al., and examined the correlations between the histological scores and the number of each population of liver-infiltrating cells. Moreover, we analyzed the surface markers of isolated liverinfiltrating cells by flow cytometry with antibodies against CD3, CD56, CD11b, lineage markers, HLA-DR, and CD1d in 5 cases. In addition, localization of CD1d expression was also examined, and analyzed the association with that of CD56 + cells by an immunohistochemical method. Results: Among various populations, only the numbers of CD56 + cells were significantly higher in NASH than those in FL (NASH: FL = 57.8 F 48.5: 15.4 F 15.2, P = 0.02). In NASH, the numbers of CD56 + cells tended to be decreased as fibrosis progresses (r = -0.55, P = 0.039), and the expression of HNE or the number of CD68 + cells showed a positive correlation with that of CD56 + cells (HNE; r = 0.56, P = 0.036, CD68; r = 0.55, P = 0.041). Moreover, flow cytometric analysis showed that NKT cells (CD3 + CD56 + ), rather than NK cells (CD3-CD56 + ), are increased in the livers with NASH. Furthermore, flow cytometric analysis showed that CD1d molecules are found to be expressed on antigen presenting cells such as macrophages (CD11b + ) and dendritic cells (lineage-DR + ). In addition, CD1d expression was significantly increased in the liver with NASH compared with FL or normal livers. Importantly, a part of CD1d expression was recognized around CD56 + cells. Conclusion: We showed that NKT cells are proliferated and activated in an early stage of NASH. In the liver with NASH, hepatocytes contain a lot of fat with the lipid being peroxidated, which generates oxidative stress. Antigen presenting cells might uptake peroxidated lipid derived from degenerated hepatocytes, and present processed lipid antigen on CD1d. NKT cells would recognize those lipid antigens through CD1d, leading to the release of various cytokines. Type 1 diabetes is in most cases the result of cell-mediated autodestruction of pancreatic h-cells. We have shown previously that DNA coding for the pro-apoptotic BAX protein promotes diabetes prevention in pre-diabetic NOD mice when co-delivered intramuscularly with DNA coding for a secreted form of the h-cell antigen glutamic acid decarboxylase (GAD). Furthermore, codelivery of BAX recruits dendritic cells containing plasmid-encoded protein in peripheral lymphoid organs. Here, we report that the same DNA vaccine reverses new onset diabetes when delivered intradermally and induces immunosuppressive CD4 + CD25 + regulatory T cells (Treg). A variety of responses to the treatment were observed. Mice responded either to both the initial and relapse treatment, to the initial treatment only, or not at all. Fifty percent of the treated mice were overtly diabetic (fasting blood glucose N 300 mg/dL) by the age of 40 weeks. By contrast, more than 90% of untreated mice and of mice treated with plasmid vector alone were overtly diabetic by that age. Immunological analysis revealed that draining lymph nodes of mice treated with the pro-apoptotic DNA vaccine contained higher numbers of Treg with enhanced immunosuppressive function compared to control animals. Our results indicate that delivery of a DNA vaccine alone can reverse the symptoms of an autoimmune disease, and suggest a real clinical potential for pro-apoptotic DNA vaccines in the treatment of immune-mediated inflammatory disorders. Recent studies concerning h cell turnover suggest that there is a continual process of h cell death and renewal. The effects of autoimmunity on this process have not been well studied but the regenerative potential of islet cells has been suggested by recent studies in animal models. We have examined changes in h cell mass and replication in the presence of islet inflammation, and during tolerance induction with CD4+CD25+ regulatory T cells.The mean percentage of replicating h cells was higher in female pre-diabetic NOD mice (age = 9-14 wks) compared to aged matched NOD/Scid mice (1.62 F 0.9, n = 6 vs. 0.19 F 0.1%, n = 5; P b 0.01). There was no significant difference in h cell mass between the two groups (0.07 F 0.04 mg, n = 4 vs. 0.09 F 0.05, n = 4; P = NS). Upon diabetes onset in female NOD mice, the percentage of replicating h cells increased to 3.56 F 0.9%, n = 3. These data are consistent with a compensatory response to inflammation.To further demonstrate that the increase in h cell replication was in response to the anti-islet autoimmune process, h cell replication was measured in NOD/Scid mice 2 and 4-5 weeks post-transfer of diabetogenic splenocytes. Our data show an increase in the mean percentage of replicating h cells with time (0.90 F 0.1%, n = 2, and 1.93 F 0.4%, n = 3; P b 0.05 at 2 and 4-5 weeks post transfer, respectively). In contrast, no change in h cell replication was seen in non-treated age-matched NOD/Scid mice (0.26 F 0.1%, n = 2 and 0.15 F 0.1%, n = 3; P = NS).Cotransfer of CD4+CD25+ regulatory T cells (Tregs), harvested from NOD mice treated with anti-CD3 mAb, prevented the development of diabetes in NOD/Scid mice by adoptive transfer of diabetogenic splenocytes. Four weeks after adoptive transfer of diabetogenic cells and Tregs, only 1 out of 5 NOD/Scid mice developed diabetes. In contrast, 5 out of 5 NOD/Scid mice that received CD4+CD25-T cells and diabetogenic cells developed diabetes (P = 0.05). In mice protected by Tregs, h cell mass was significantly greater than in those that received CD4+CD25-cells (0.02 F 0.02 mg, n = 5 vs. 0.00011 F 0.00001 mg, n = 5, respectively; P = 0.03). The percent of replicating h cells in mice that received Tregs was 1.97 F 0.6%; n = 5, whereas h cell mass was too small to determine replication rates in recipients of CD4+CD25-cells.These findings suggest that in NOD mice, islet inflammatory lesions can play a role in stimulating h cell replication. Nevertheless, heightened h cell replication, initially in response to autoimmunity, is insufficient to overcome the rate of autoimmune-mediated beta cell destruction. Treatment with Tregs leads to improved preservation of h cell mass with maintenance of heightened h cell replication and prevents the development of diabetes in NOD mice. The majority of female NOD mouse develops immune mediated diabetes between 15 and 35 weeks of age but a subset remain non-diabetic after 40 weeks of age. Lack of diabetes development in these mice could be due to a failure of the immune system to completely target and destroy beta cells or to the presence of insulin producing cells resistant to destruction or both. In order to distinguish between these two hypotheses, we studied the pancreatic histology of 11 non-diabetic female NOD mice that remained non-diabetic after 40 weeks of age. Each of the eleven mice had severe islet lymphocytic infiltrates, which affected the majority of the islets. In 5/11 mice there was no insulin staining while in 5 mice there were islet cells that exhibited double positivity for insulin and glucagon. In one mouse, we could detect both single insulin positive cells (negative for glucagon) and cells with double positivity. In all mice studied, glucagon positive cells were preserved and in mice with insulin positivity only a subset of the glucagon positive cells expressed positivity for insulin. Transfer of diabetes was analyzed for six of these NOD mice by injecting 3 Â 10 7 splenocytes in 8-10 week old SCID-NOD recipients. These splenocytes rapidly induced diabetes (b 5 weeks).These data show that older non-diabetic NOD females show insulitis with loss of beta cells and that their splenocytes are capable of transferring diabetes. Furthermore, the surviving insulin positive cells show an unusual phenotype characterized by positivity for glucagon. Further studies will elucidate the origin of these cells and we hypothesized that double positive cells resist immune mediated beta cell destruction though alternatively they may represent unusual recently developed insulin expressing cells. We observed that B cell deficient NOD mice, which are normally resistant to diabetes, develop the disease following PDL1 blockade whereas CD4 deficient NOD mice were resistant to PDL1 blockade; thereby suggesting CD4+ T cells play an important role in anti-PDL1 mediated acceleration of diabetes. We then utilized BDC2.5 TCR Tg mice to more clearly dissect the role of PDL1 in regulating autoreactive CD4+ T cells. PDL1 blockade precipitated early onset diabetes, an expansion of BDC2.5 TCR Tg cells, and decreased apoptosis. NOD congenic mice that have a variable degree of resistance to autoimmune diabetes (NOD.Idd5, NOD.Idd3, NOD.Idd3/10/18, and NOD.Idd9 usually develop diabetes with a incidence of 40%, 20%, 8% and 3%, respectively) were then utilized to study the role of PDL1 in diabetes resistance. Anti-PDL1 mAb treatment accelerated diabetes in all the congenic strains albeit with different acceleration patterns. Interestingly, in NOD.Idd9 congenic mice, PDL1 blockade both accelerated the time of onset as well as increased the incidence of diabetes to 50%. Utilizing Idd9 mice, we have shown that blockade of PDL1 results in precipitation of diabetes by changing the local cytokine milieu from Th2 to Th1 cells, with upregulation of IFN-gamma and TNFalpha. We conclude that PDL1 regulates autoimmune diabetes by limiting the expansion of autoreactive Th1 cells and thus plays a critical role in mediating disease resistance. These results provide the rationale for developing novel therapies to re-establish tolerance in autoimmune diabetes. Type 1 diabetes is an immune-mediated disease characterized by the autoimmune destruction of pancreatic h-cells. We have previously developed an experimental autoimmune diabetes (EAD) model with insulin peptide B:9-23 immunization and/or polyinosinic-polycytidylic acid (PolyI:C), as a viral RNA mimic Toll-like receptor (TLR) activator, in transgenic mice expressing the costimulatory molecule B7.1 in their islets (drivenly the Rat Insulin Promotor, RIP). A major goal is the development of bhumanizedQ mice that can develop immune mediated diabetes with known native autoantigen stimulus. We have combined transgenes for human A2.1 with B7.1 transgenic (BALB/c B7.1x C57Bl/6 A2.1 mice, original C57Bl/6 B7.1 mice were a gift from Dr L. Wen and C57Bl/6 A2.1 mice were provided from Dr B.L. Mice were immunized with B:9-23 with or without PolyI:C. Overall 29% of the mice by 41 weeks (n = 7, range diabetes onset 22-43 weeks) developed diabetes following Poly:IC injection alone. Forty-three percent developed diabetes by 26 weeks (n = 7; range 21-26 weeks, P b 0.54) with B:9-23 immunization and PolyI:C injection. Our studies indicate that human A2 is permissive for the development of diabetes in EAD but does not accelerate disease in the model following either TLR3 activation or peptide B:9-23 immunization. Studies are underway to define potential islet peptide A2 restricted antigen presentation using ELISPOT analysis in this model of immune mediated h-cells destruction.Sa1.134. Similarities and Differences in Autoimmune Responses between Type 1 and Type 1.5 Diabetes Patients. Type 1.5 diabetes or LADA comprises approximately 10% of Caucasian adult phenotypic type 2 diabetes patients. The islet reactive autoantibodies and T cells found in type 1.5 diabetes patients suggest an autoimmune pathogenesis. In this study, we asked how T cell reactivity and phenotype compared in type 1.5 versus type 1 diabetes. We identified type 1.5 diabetes patients (n = 12) through autoantibody and T cell responses to islet proteins. T cell reactivity to islet proteins was measured by cellular immunoblotting and peripheral T cell populations were measured by FACS and compared between type 1.5 patients versus type 1, type 2, and normal controls. T cells from both type 1 and type 1.5 diabetes patients respond similarly to islet proteins in the molecular weight regions of 116kDa, 97kDa, 60kD. In contrast, islet proteins in the molecular weight regions of 65-90kDa and 21-38kDa were significantly (P b 0.05) less stimulatory to T cell responses from type 1.5 diabetes patients versus type 1 patients. The number of CD4+CD25+ cells was significantly lower (P b 0.05) in the peripheral blood of type 1 patients compared to type 1.5 patients, type 2 patients and normal controls. CD4+CD38+ cells were significantly lower in the peripheral blood of type 1.5 patients compared to the other subject groups and T cell receptor positive gd cells were significantly (P b 0.05) decreased in the peripheral blood of type 1 and type 1.5 patients. These results suggest that type 1 and type 1.5 diabetes are immunologically similar in some respects but in other ways are different. These differences may be important in the slower disease progression of the type 1.5 diabetes disease process. Several viral infections including cytomegalovirus (CMV) infections have been implicated to be associated with the development of type 1 diabetes-related autoimmunity.The aim of this study was to explore whether there is any correlation between CMV infections and type 1 diabetes-associated autoimmunity in young children with HLA-conferred disease susceptibility. The cases with diabetes-associated autoantibodies and their HLA, age and sex-matched controls were participants in the Diabetes Prediction and Prevention (DIPP) Study running at the Universities of Turku, Tampere and Oulu in Finland. According to the study protocol, newborn infants carrying susceptible HLA genotypes are observed at 3 to 6 months intervals and regularly tested primarily for ICA and if positive also for GADA, IA-2A and IAA. Forty-one prospectively followed antoantibody positive subjects (21 girls, 20 boys, age 3 to 48 months, median 18 months) and 190 sex, age and HLA-matched controls were analyzed for CMV IgG class antibodies by EIA at the time of seroconversion to autoantibody positivity or within the next 6 months. No significant difference was seen in the prevalence of CMV antibodies. At the time of seroconversion 10 (24%) of the autoantibody positive subjects and 60 (32%) of their controls were positive for CMV IgG class antibodies (P = 0.47, Chi-square test). No differences were either found between ICA, GADA, IAA or IA-2 positive subjects and control subjects when each autoantibody was analyzed separately.In conclusion, these data suggest that early cytomegalovirus infections do not increase the probability to develop type 1 diabetes-associated autoimmunity. Rotavirus infections have been implicated as a possible trigger of T1D. We elucidated this connection by comparing peripheral blood T-cell responses to rotavirus between children with newly diagnosed T1D (n = 37), children with T1D-related autoantibodies (n = 34) and control children carrying HLA-conferred susceptibility to T1D but without T1D-related autoantibodies (n = 104). Lymphocyte proliferation assays based on stimulation with an antigen were performed using freshly isolated peripheral blood mononuclear cells. We measured also childrenTs IgG and IgA class rotavirus antibodies in plasma samples drawn at the same time as the T-cell samples. No differences were observed in the strength of T-cell responses to rotavirus between the children with overt T1D or multiple autoantibodies, or the control children. Furthermore, also the frequencies of positive T-cell responses to rotavirus were closely similar in all three groups. The result remained similar, when only the children with serological evidence of earlier rotavirus infections were studied. Further, no differences were observed in the responses to the control antigens PPD and tetanus toxoid, but T-cell responses to purified coxsackie B4 virus were stronger in the children with autoantibodies than in the control children. In conclusion, our cellular immunity studies provided no evidence supporting association of rotavirus infections with T1D or presence of T1D-related autoantibodies in young children. Diabetes is associated with infringment in endogenous opioid system and opposite alterations in various type opioid receptor sensitivity. However, it is not clear whether membrane lipid modification processes take part in development of such disorder as well, as their possible role in etiology of neurological complications in these patients. In order to evaluate processes mentioned involvement we suppose to investigate effects of selective delta-opioid receptor agonist Deltorphine II, delta-1receptor antagonist 7-Benzylidennaltrexone and delta-2 receptor antagonist Naltriben on some aspects of phosphoinositide cycle functioning in lymphocytes. The regulator role of processes mentioned at the initial membrane-bound step of studied opioid agonist and antagonists information translocation has been demonstrated in both diabetic and non-diabetic cells. The evidence exists that alterations in lipid-mediated signal transduction pathways and receptors lipid microenvironment may be responsible for altered nociception at diabetes conditions. However, it is not clear whether membrane lipid free radical oxidation processes and enzymes of antiradical defense system take part in such disorders. In order to evaluate the involvement of above mentioned processes in altered nociception at diabetes conditions the intensity of lipid peroxidation processes have investigated as well, as activity of enzymes of cell antiradical protection, superoxid glutathione reductase dismutase in diabetic patients erythrocyte membranes at the conditions of opioid receptors blockade and sensitization. Some correlation between intensity of enzymatic lipid peroxidation and opposite changes of receptor sensitivity as well. as possible participation of cell antiradical protection enzymes have been demonstrated. The evidence exists, that studied lipid mediated transduction pathway together with receptor microenvironment modifications appear to be possible biochemical targets of altered nociception at diabetes conditions. Rationale: A central issue when using immune-suppressive therapy in transplantation and autoimmunity is to balance benefits with systemic side effects. Administration of non Fc-binding anti-CD3 has shown good efficacy in temporarily maintaining Cpeptide levels in recent-onset diabetes over a 1-2 year timeframe, but immunosuppressive side effects limit higher-dose regimens or continuous administration. One novel attractive avenue in type 1 diabetes (T1D) is the islet autoantigen-specific induction of adaptive regulatory T cells (Tregs) that can act in vivo as bystander suppressors of autoaggressive responses selectively within the pancreatic draining lymph node (PDLN). In our hands, such cells can be induced via oral or intranasal administration of insulin, proinsulin peptides and DNA vaccines expressing islet antigens and efficacy is associated with IL-4 and IL-10 production. Since anti-CD3 is known to increase numbers of intrinsic CD25+ Tregs as well as TGF-h and IL-10 production, we reasoned that it would create a favorable systemic milieu for islet-antigen specific induction of autoreactive, adaptive Tregs and synergy could be expected.Approach: Combinatorial therapy of recent-onset diabetes in NOD and RIP-LCMV mouse models using anti-CD3 FabT 2 and intranasal proinsulin.Results: At least 50% increased reversion of recent-onset diabetes was observed in NOD and RIP-NP mice treated with a combination of anti-CD3 and proinsulin compared to each intervention given alone. In particular, mice with more rapid beta cell loss benefited from this synergistic effect. Mechanistically, we observed increased numbers of proinsulin specific antigen-induced Tregs producing IL-4 and IL-10 in mice that received combinatorial therapy compared to those receiving anti-CD3 alone. Furthermore, we found much higher numbers of TGF-beta producing CD25+ Tregs in mice treated with both, compared to mice that received anti-CD3 alone.Conclusion: Antigen-specific induction of Tregs can synergize with systemic immune modulatory approaches to revert recentonset T1D. Synergy was evidenced by enhanced clinical efficacy and increased numbers of intrinsic TGF-beta producing CD4+CD25+ as well as adaptive autoantigen-specific IL-4/IL-10 producing Treg populations. This strategy should be considered for the clinic and maybe also for transplantation. is supported by a JDRF postdoctoral fellowship and MGVH is supported by DK51091, AI44451 and a U-19 prevention center grant. CD4 + CD25 + FoxP3 + T cells are thought to be essential in maintaining tolerance to self antigens. These cells, known as regulatory T cells (T R ), have been classified in two main categories: natural T R, which are derived from the thymus, and adaptive T R , which arise in the periphery. The importance of these cells in controlling responses to foreign antigens has just begun to be described. Previously, we have reported that activation of human CD4 + CD25-T cells led to expression of FoxP3 in CD25 + cells and acquisition of cell contact-dependent, cytokine-independent regulatory activity. Results in our laboratory reveal that these in-vitro derived T R could be generated from both memory and naive cells and by activation of CD4 + CD25-T cells with alloantigen or a foreign antigen, HA (307-319). In the HA system, MHC class II tetramers were used to identify antigen-specific T cells. Antigen activation of CD4 + CD25-led to two populations of CD4 + CD25 + cells, Tetramer + and Tetramer-cells, with antigen specific suppression occurring only in the Tetramer+ cells. HA generated T R required cognate antigen for activation, but once activated subsequently suppressed noncognate bystander T cell responses as well. For this reason, we have started testing the ability of a diabetes autoantigen, GAD65, to generate antigen-specific T R. GADspecific T R can be generated from both normal and diabetic subjects, and once activated these T R also suppress bystander T cell responses. This raises the possibility that antigen-specific T R may be useful therapeutically in autoimmune diseases by localizing generalized suppressive activity to tissues expressing select target antigens. TGF-h is produced by a variety of cell types and can exhibit strong immune dampening functions. Surprisingly, in a model of virally-induced autoimmune diabetes, local expression of TGF-h in h-cells under the control of a doxycycline-dependent promoter resulted in increased islet infiltration and disease incidence. This was attributed to a selective anti-apoptotic effect of TGF-h on differentiated memory-effector CTL. Conversely, mice lacking functional TGFh-receptors on T cells exhibited enhanced apoptosis and fewer memory CD8 lymphocytes. The effects of TGF-h were clearly dependent on the T cell differentiation status, as it effectively suppressed activation of naRve anti-viral CTL. Our results highlight a novel aspect of the pleiotropic nature of TGF-h, and have implications for the design of immuno-therapies involving this cytokine. Invariant natural killer T (iNKT) cells comprise a subset of regulatory T cells characterized by their co-expression of NK cell markers and an invariant TCR that recognizes a lipid antigen in a CD1d-restricted manner. Previously, we reported that activation of iNKT cells by the sphingoglycolipid alphagalactosylceramide (a-GalCer) protects against type 1 diabetes (T1D) in NOD mice. This protection involves the polarization towards a Th2-like immune response accompanied by increased IL-4. As potent activation of iNKT cells also trans-activates other immune cells, we further analyzed whether iNKT cells influence the function of CD4+CD25+ regulatory T cells. We found that CD4+CD25+ T cells from NOD.CD1d-/-mice deficient in iNKT cells functioned similarly in vitro to CD4+CD25+ T cells from wild-type NOD mice and suppressed the proliferation of NOD T responder cells upon stimulation with a-GalCer. Adoptive transfer of NOD diabetogenic T cells and NOD CD4+CD25+ T cells pretreated in vivo with multiple low doses of a-GalCer further indicated that activation of iNKT cells do not influence the regulatory capacity of CD4+CD25+ T cells to inhibit the transfer of T1D. In contrast, protection from T1D mediated by adoptive transfer of activated iNKT cells requires the presence of CD4+CD25+ T cells, as splenocytes pretreated with a-GalCer and depleted of CD25+ cells were unable to confer protection from T1D. These data suggest that even though iNKT cells may not influence CD4+CD25+ T cell activity, iNKT cell-mediated protection appears to require the presence of CD4+CD25+ T cells. NBI-6024 is an altered peptide ligand (APL) corresponding to the 9-23 amino acid region of the insulin B chain (B (9-23) ), which is an epitope recognized by interferon (IFN)-g-producing T lymphocytes in type 1 diabetic patients. Immunomodulatory effects of NBI-6024 in recent-onset type 1 diabetic patients in a phase I clinical trial (NBI-6024-0003) were measured using the ELISPOT assay in peripheral blood mononuclear cells. IFN-g responses to B (9-23) were observed in 62.5% (5 of 8) of patients receiving placebo and in only 8% (1 of 13) of non-diabetic untreated control subjects. NBI-6024 administration (five biweekly or biweekly-monthly s.c. injections) led to a dose-dependent reduction in the percentage of patients with IFN-g responses to B (9-23) and a concomitant increase in the percentage with IL-5 responses to B (9-23) . Similar trends were observed with NBI-6024specific ELISPOT responses. This Phase I clinical study demonstrated that NBI-6024 treatment did not enhance, but rather suppressed, the pathogenic IFN-g response of recent-onset type 1 diabetic patients in a dose-dependent fashion. Enhanced IL-5 responsiveness after NBI-6024 treatment is consistent with induction of a T helper (Th) 2-like immunological phenotype. The significance of these findings on the clinical outcome of disease is under investigation in a Phase II multi-dose study. There is an excess of HLA-DR3/DR4 heterozygous individuals among Caucasian patients with T1D. The incidence of T1D is rising in many countries and the MHC haplotype composition of T1D patients has also changed over the past several decades. Earlier, we proposed that, for any polygenic disease, if there is mixing of two populations with reciprocally different frequencies of susceptibility genes at different loci, the incidence in the mixed offspring will be much higher than in the original populations because of susceptibility gene complementation at those different loci. We propose that the apparent increased risk for HLA-DR3/ DR4 heterozygotes as compared with homozygotes for either HLA-DR3 or DR4 and the heterozygote overrepresentation among patients is because their parents appear to come from different, previously isolated populations. Thus, HLA-DR3 and DR4 are both disease and population markers. Our evidence is that (a) HLA-DR parental genotype distribution deviated from the Hardy-Weinberg equilibrium in a way opposite to the patients: the parents had a paucity of HLA-DR3/DR4 heterozygotes, (b) DR3-transmitting parents had different HLA-A2 frequencies on their untransmitted HLA haplotypes than those who transmitted DR4, (c) DR3-positive parents had different INS allele frequencies than DR4-positive parents, and (d) parents of T1D patients had greater self-reported ethnic mixing than control parents. Thus, a specific kind of intrafamilial population admixture explains all three phenomena. Concordance for T1D is approximately 40% for monozygotic twins, approximately 12% for MHC-identical sibs, and approximately 6% for sibs in general. Thus, there is an MHC T1D susceptibility gene, but non-MHC genes are also required for susceptibility. From the population distribution of certain MHC markers and from analysis of affected sib pairs, the MHC susceptibility gene(s)must be expressed recessively and have a frequency in the general population of approximately 0.53. Because all of the MHC markers for T1D are embedded within conserved extended haplotypes with fixed DNA over at least the HLA-B to HLA-DRB1/-DQB1 interval (around 1 megabase in length), identification of the true T1D susceptibility locus has been difficult. The presence at low frequency of so-called protective HLA-DR/DQ haplotypes in patients strongly suggests that HLA-DRB1*0301, DQB1*0201 (DR3) and HLA-DRB1*04, DQB1*0302 (DR4) are markers for susceptibility but not themselves the true MHC susceptibility gene(s), which remain(s) to be discovered. Intrinsic penetrance is the concordance rate in monozygotic twins and is the rate at which all completely susceptible persons in the population (who have all necessary susceptibility genes) have T1D. Penetrance of susceptibility genes is not likely due to differential environmental effects since the concordance rate in dizygotic twins is the same as the rate in all sibs. From the fact that homozygotes for MHCdetermined dominantly expressed IgD deficiency have about twice the frequency of the deficiency as heterozygotes, we concluded that penetrance in that situation is stochastic and a property of the MHC susceptibility gene. It seems likely that a similar mechanism is operative in T1D. The rare X-linked syndrome known as IPEX (Immune dysregulation, Polyendocrinopathy, Enteropathy and X-linked inheritance) is characterized by early-onset IDDM (type 1 diabetes), severe enteropathy, eczema, and variable autoimmune phenomena. We have observed the IPEX phenotype in one patient, at the age of one month, with intractable diarrhea, dermatitis and elevated IgE. Autoantibodies against pancreas, thyroid and gut were negative. The mutations consist of a known splice-site mutation (exon 4 and 5 boundary), and two other aberrations (within intron 8 and the second within exon 9), previously not described. Interestingly, expression studies of FOXP3 mRNA detected a product of about 100bp smaller compared to the normal control. Molecular analysis extended to other members of the family showed the same FOXP3 mutations in one of the two healthy brothers of the patient; in addition the mother was identified as a carrier for the same mutations. At present, the patient is 17 months old and his diarrhea is under remission without treatment, as he is routinely monitored in order to control the disease progression. IPEX is a syndrome usually associated with overwhelming autoimmunity and severe phenotype. Here we present an atypical case of IPEX manifesting in unusually mild clinical features corresponding to a novel set of FOXP3 mutations. Further studies will be important to clarify the link between the phenotype of this patient and the specific molecular aberrations in the FOXP3 gene. Moreover, whether the gene product without FOXP3 exon 2 is a normal splicing variant of FOXP3 mRNA, or is the result of multiple mutations found in the patient is under investigation. Purpose: KL-6 is a human glycoprotein secreted by type II alveolar cells in lung, and its serum levels increase in pneumonia of various causes. KL-6 is a member of the MUC-1 family, which is expressed in cornea and conjunctiva as well as lung. The purpose of the present study is to investigate the clinical usefulness of quantifying serum KL-6 levels for diagnosing and following up sarcoidosis in patients with uveitis. Immunology of the Eye Patients & Methods: Sera were obtained from 24 uveitis patients diagnosed as sarcoidosis, 37 uveitis patients with other etiologies, and 138 healthy control subjects. Patients were considered to have indication for sarcoidosis when their KL-6 concentrations exceeded 370 U/ml. Within this criterion, 137 of 138 (N99%) of healthy subjects were negative for sarcoidosis. Serum KL-6 concentration was determined by a human KL-6 electrochemiluminescence immunoassay (ECLIA).Results: The average level of KL-6 in sera of uveitis patients with sarcoidosis, other etiologies, and healthy controls were 387 F 52 (Mean F SD) U/ml, 266 F 23, and 183 F 6, respectively. The level of KL-6 in uveitis patients with sarcoidosis was significantly higher than in patients with other etiologies of uveitis. When the KL-6 results were combined with serum angiotensinconverting enzyme (ACE) concentrations, 87.5 % of sarcoidosis patients were identified, compared to 66.7 % using ACE results alone. And there were significant correlations between serum KL-6 and ACE levels in the patients with sarcoidosis. Moreover, serum KL-6 concentrations were less affected by systemic corticosteroid administration than ACE, and never affected by ACE inhibitory drugs for systemic hypertension.Conclusions: Combined measurements of serum KL-6 and ACE may be useful as a screening for sarcoidosis in uveitic patients. And also, it may be valuable to follow up the diagnosed sarcoidosis, because the concentration of serum KL-6 less fluctuates than that of ACE in patients treated with systemic corticosteroids and/or anti-hypertensive drugs.Supported by JSPS Research Fellowship for Young Scientists, Japan Society for the Promotion of Science, Tokyo.Sa2.126. The Involvement of Autoimmunity Against Retinal Antigens in Determining Disease Severity in Toxoplasmosis.A. 1 1 Immunology, University of Sao Paulo, Sao Paulo, São Paulo, Brazil; 2 Ophthalmology, Federal University of Sao Paulo, Sao Paulo, São Paulo, Brazil.PURPOSE. Ocular lesions are frequent in various individuals infected with Toxoplasma gondii. Immunologic parameters in the response to retina antigens were evaluated in infected persons with and without ocular lesions and in no infected controls. Subjects were divided into groups on the basis of titers of serum antibodies to T. gondii, presence and severity of ocular lesions, and clinical history.RESULTS. Peripheral blood mononuclear cells from patients with mild disease responded to one or more retinal antigens with a significantly higher frequency than patients without disease or with severe disease. Interestingly, the cytokines produced by the proliferating mononuclear cells did not follow any specific patters, except for the fact that IL-4 and IL-5 were seldom detected.CONCLUSIONS. Our results suggest that although the presence of an immune response towards autoantigens is not protective against the development of ocular lesions by the Toxoplasma gondii, it may protect against the development of severe disease. Cytotoxic CD8 bright CD56+ T Cells are Immunopathogenic Effectors in Patients with Active BehcetTs Uveitis. 1 1 Ophthalmology, Seoul National University College of Medicine, Seoul, Seoul, Republic of Korea.Recent our report demonstrated that the intraocular infiltration of CD8 bright CD56+ T cells was a distinct feature in active BehcetTs uveitis (BD) from other etiologies of endogenous uveitis. However, the phenotypic natures and effector functions of CD8 bright CD56+ T cells in active BD have remained elusive. This study was conducted to determine the phenotypic and functional characteristics of CD8 bright CD56+ T cells and to investigate the cytotoxic mechanisms of theses subsets. Forty five patients with BD (active: 24, inactive: 21) and 20 healthy controls were recruited in this study. Phenotypic analysis of fresh PBMCs were performed using anti-CD8 mAb and anti-CD56 mAb in conjunction with a three-or four-color immunofluorescence tests for the expression levels of the following molecules: CD11b, CD27, CD45RA, CD45RO, CD62L, CD94, NKG2D, HLA-DR. Flow cytometric measurements of intracellular cytokines (IFN-g and IL-4) and cytotoxic molecules (intracellular perforin and surface FasL) were performed by in vitro PMA and ionomycin (PI) stimulation. Ex vivo cytolytic capacities of purified CD8 bright CD56+ T cells against K562, Raji, and human umbilical vein endothelial cell line (HUVEC) were measured by standard 51 Cr release assay. Modulation of cytotoxicity was done using the treatment of HUVEC by rhIFN-g and the treatment of effector cells by concanamycin A (CMA) or brefeldin A (BFA). CD27 and CD62L were down-regulated on peripheral CD8 bright CD56+ T cells in patients with active BD in contrast to the up-regulation of CD11b and HLA-DR. Interestingly, CD94/ NKG2A was up-regulated on peripheral CD8 bright CD56+ T cells in BD in contrast to the down-regulation of NKG2D. Furthermore, in patients with active uveitis, these subsets were polarized to produce IFN-g, contained high amounts of preformed intracellular perforin, and exclusively expressed surface FasL upon stimulation by PI. Moreover, in vitro cytolytic functions of CD8 bright CD56+ T cells in active BD were up-regulated against both K562 and Raji, which were effectively inhibited by CMA. Interestingly, in vitro cytolytic activity of these subsets against HUVEC was also up-regulated, which was effectively suppressed by BFA rather than by CMA. Cytolytic functions of PI-stimulated CD8 bright CD56+ T cells were greatly enhanced against HUVEC, which was augmented by pretreatment of IFN-g on HUVEC. CD8 bright CD56+ T cells, characterized by cytotoxic effector memory Tc1 phenotypes with functional NK receptors, play immunopathogenic roles in BD and exhibit strong cytolytic functions against vascular endothelial cells through FasL-dependent pathway. Experimental autoimmune uveitis (EAU), a model for human uveitis, is induced in mice by immunization with a retinal antigen in complete FreundTs adjuvant and pertussis toxin. Here we describe a new EAU model induced with in vitro-matured, retinal antigen-pulsed DC. DC were expanded in vivo by hydrodynamic injection of 50 Ag of Flt-3 ligand DNA. On day 5, the size of the spleen was doubled and the CD11c+ population tripled from an average of 4.9% to 15.0%, for a total increase of 6-fold over background. When pulsed with a uveitogenic peptide, these cells induced vigorous immune responses in vivo. Furthermore, 2 injections of DC 4 days apart plus pertussis toxin elicited a typical EAU-like inflammation in eyes of susceptible B10RIII mice, with an incidence of up to 87%. Sorted CD8a+ and CD8a-DC subpopulations exhibited differential cytokine production when stimulated as above, with the CD8a-population releasing more IL-1h, IL-2, IL-6, IL-10 and TNF-a than the CD8a+ population, whereas CD8a+ DC produced more IL-12 and IFN-g. Current work is aimed at examining differences in the ability of these splenic DC subpopulations to induce EAU. This alternative EAU model, which allows direct manipulation of DC in vitro, will permit better characterization of the role of these cells in autoimmunity to the retina and may help to devise new approaches to therapy. Soluble factors released by cultured RPE cells were previously shown to suppress T cell proliferation, and to confer T cells with regulatory properties. This study was performed to investigate the possibility that molecules on the surface of RPE cells can exert similar effects on T cell function. T cells activated via soluble a-CD3 were cultured in the presence or absence of fixed RPE cells for 72h in serum free medium, and their proliferation, as well as their ability to suppress the proliferation of bystander T cells, was assessed via 3H thymidine incorporation. For the latter, T cells were irradiated at the end of the 72h co-culture period with RPE cells, and were then washed and re-plated with freshly isolated T cells activated with a-CD3 antibodies. T cell viability was quantified using the alamar blue bioassay. In order to investigate whether surface TSP-1 played a role in mediating the contactdependent effect of RPE cells on T cell function, the above experiments were repeated using fixed RPE cells recovered from the eyes of TSP-1 knockout mice. These studies were complemented with immunohistochemistry and western blotting in order to confirm the expression of TSP-1 by cultured RPE cells. Finally, to begin to address the potential TSP-1 receptors on T cells involved in mediating this effect, we examined the importance of the integrin a4h1 (VLA-4), used by naive and activated T cells for attachment to the TSP-1 molecule. This was done by preventing the binding of TSP-1, on RPE cells, to VLA-4 on T cells, with blocking antibodies, and by blocking the function of its associated potassium channel, Kv1.3, with margatoxin (MgTx). Our results show that fixed RPE cells suppress the proliferation of activated T cells, and in turn confer them with a similar ability to suppress bystander proliferation. Further, cultured RPE cells expressed TSP-1 homogeneously throughout their cell surface, and fixed RPE cells lacking the TSP-1 gene, failed to suppress T cell proliferation. Similarly, inhibiting TSP-1 binding to its receptor VLA-4 on T cells, or blocking the function VLA-4Vs associated potassium channel, Kv1.3, prevented T cells from becoming regulatory. We conclude that aside from soluble factors, RPE cells also exhibit a contact-dependent mechanism, mediated by TSP-1/VLA-4 interactions, by which they suppress the proliferation of activated T cells, and confer them with regulatory properties. Altered Peptide Ligands of a Retinal Antigen Protect from Anti-Retinal Autoimmunity by Eliciting Active Regulatory Mechanisms.L. M. Cortes, 1 D. Avichezer, 1 P. B. Silver, 1 C. C. Chan, 1 R. R. Caspi. National Eye Institute, National Institutes of Health, Bethesda, MD, USA.We identified altered peptide ligands (APL), capable of immunomodulating experimental autoimmune uveitis (EAU), a Th1-driven disease induced in B10.RIII mice by immunization with the retinal antigen IRBP in complete FreundTs adjuvant. Alanine-substituted peptides of the major pathogenic epitope, residues 161-180, were synthesized. They were tested for immunogenicity, crossreactivity with the native 161-180 epitope, pathogenicity, and ability to prevent EAU when given in incomplete FreundTs adjuvant (IFA) 2 weeks before EAU challenge with native p161-180. Two peptides, 169A and 171A, were unable to elicit disease and crossreacted with p161-180 by lymphocyte proliferation. Mice pre-treated with either of the putative APL failed to develop EAU and had reduced cellular responses to p161-180 by lymphocyte proliferation and by delayed hypersensitivity. Their cytokine response profile to p161-180 showed reduced IFN-g and enhanced IL-4, and serum antibody titers to p161-180 revealed reduced IgG2a and elevated IgG1 isotypes, suggesting a Th2 shift in the response. Protection was transferable with lymphoid cells from protected donors to naRve recipients who were subsequently immunized for EAU. Thus, APL pretreatment appears to prevent induction of EAU by skewing the subsequent response towards a non-pathogenic effector phenotype, as well as by eliciting regulatory cells. Introduction: Granulocyteapheresis (GCAP) is a novel treatment that has the capability of modulating innate immune response in some autoimmune diseases such as Ulcerative Colitis, Crohn's Disease, Rheumathoid Arthritis, and Behcet's Disease. We present our results with this treatment in a period of 1 year in 3 cases of ocular Behcet's Disease.Objective: to evaluate GCAP efficacy to control uveitis activity, number of relapses and steroid sparing effect in a 1 year period.Material and Methods: 3 patients with ocular Behcet disease resistant to conventional medical treatment were selected for GCAP treatment. One patient presented active uveítis and the others were clinically inactive. Patients demographics were: age 25,7 F 3,1;2 female/1 male; time from diagnose 66,7 F 45,7 months; average of uveítis relapses/year 5,38 F 3.6; all patients were steroid dependant and needed cyclosporine A (2/3), azathioprine (2/3) and/or micophenolate mofetil (1/3) to control the disease. We performed an induction treatment of 1 GCAP session/week during 10 consecutive weeks. Patients were maintained with 1 GCAP session each month during 1 year. The GCAP procedure consists in an extracorporeal blood circulation through a column filled with cellulose diacetate beads. Each procedure lasts 1 hour and 1.8 liters of blood are processed at 30 ml/h. Visual acuity, intraocular inflammation degree, and prednisone requirements were assessed during the study period. No adverse event were reported.Results: During induction treatment uveitis was controlled in all patients and none of them presented any relapse. Visual acuity was stabilized in 3 eyes and improved in the other 3. Prednisone dose was tapered down from 35,8 F 13.7 to 15 F 5 mg/day. After completion of 1 year maintenance treatment, mean number of uveitis relapses were 1 F 1, and daily prednisone dose was reduced to 21.6 F 7.6. Visual acuity was stable during maintenance treatment.Conclusion: GCAP treatment is a safe and effective treatment for ocular Behcets Disease refractory to conventional medical treatment. A one year maintenance regimen can reduce uveitis relapses, preserve visual acuity and reduce prednisone requirements. The eye (and especially the highly vascular tissues such as the uvea and the conjunctiva) is considered a special btargetQ for immunopathologic reactions. Uveitis refers to inflammation of the uveal tract, which includes the iris, ciliary body, and choroid. Although the aetiology is unknown in most cases, many patients have an associated underlying systemic disease. Uveitis can be the initial manifestation of an autoimmune systemic disease, and may appear years before the diagnosis of the primary disease. A retrospective study was conducted of patients with uveitis to determine the frequency of associated autoimmune systemic diseases and to assess the value of limited laboratory screening of these patients. Materials. 64 patients (33 male, 31 female) with uveitis (38 anterior, 14 posterior, 4 intermedia, 8 panuveitis) were studied. All patients underwent a standard diagnostic protocol including the following immunological tests: serum immunoglobulins, complement components, circulating immune complexes (CIC), antinuclear antibodies (ANA), antineutrophil cytoplasmic antibodies (ANCA), anticardiolipin antibodies (ACA) and major histocompatibilty complex antigens. Results: Overall 87.5% of patients had at least one detectable immunological abnormality. 14/64 patients had detectable levels of ANA (titer 1/ 40-1/320) (21.9%), 8/64 IgM ACA (12.5%), 7/64 IgG ACA (10.9%), 5/64 rised ANCA (7.8%) and 4/64 positive rheumatoid factor (6.3%). A relationship with a subclinical autoimmune systemic disorder could be presumed in 11/64 cases (17.2%) defined as the presence of autoantibodies (ANA, ANCA or ACA) in the presence of complement comsumption, hypergammaglobulinemia o increased CIC. HLA-B27-associated anterior uveitis was observed in 8/64 patients (12.5%), HLA-DR52-associated posterior uveitis in 2/64 (3.1%), HLA-DR53-associated Vogt-Koyanagui-Harada syndrome in 1/64 (1.6%) and HLA-A29associated birdshot retinochoroidopathy in 1/64 (1.6%). A definite association with a systemic autoimmune disease was determined for 8/64 patients (12.5%) most of them with lupus like disease (LLD) (4), LLD plus antiphospholipid syndrome (1), Sjfgren syndrome (2) and systemic vasculitis (1) . The presence of the systemic autoimmune disease was not suspected prior to eye involvement and was only recognised after the subsequent diagnostic procedures. In a proportion of patients with uveitis an autoimmune systemic disorder may be present. The systemic autoimmune diseases were frequently undiagnosed before the onset of the ocular disease and before the uveitis consultation. Studies of the immunological profile can therefore help in further assessment of patients with uveitis. Purpose: To evaluate the ability of computer-based algorithms (in silico) to identify putative auto-antigenic peptides and to evaluate predicted binding of putative auto-antigenic peptides in different populations with the same disease. Methods: Three internet-accessible computer-based algorithms were used to predict binding of tyrosinase (TYR) and tyrosinase-related protein-1 (TRP-1)-derived peptides to HLA-DRB1*0405. These results were compared to published studies of in vitro immunogenic responses to TYR and TRP-1-derived peptides by HLA-DRB1*0405restricted T cells from patients with Vogt-Koyanagi-Harada (VKH) disease. Three websites (SYFPEITHI, MHC-Thread, and Propred) were used to determine relative likelihood of binding scores for the immunogenic TYR and TRP-1 peptides determined in vitro to HLA molecules that confer risk for VKH disease in different populations, HLA-DRB1*0101, DRB1*0404, and DRB1*0405. Results: We found that using all three websites together, at least one in silico fragment overlapped with each of the in vitro peptides by at least nine amino acids with the exception of TRP-1 p243-254, which overlapped with a fragment predicted by Propred by seven amino acids. TYR p134-146, TYR p423-434 and TYR p426-437 were found by Propred to have a high likelihood of binding to HLA-DRB1*0101, DRB1*0404, and DRB1*0405, while TYR p193-203 and TYR p429-440 were predicted by Propred to have a high likelihood of binding only to HLA-DRB1*0405. Predicted binding of immunogenic peptides to HLA-DRB1*0101 and DRB1*0404 is consistent with clinical studies that have found that these alleles confer risk for VKH disease in Mestizo individuals. Conversely, differences between likelihood of binding of immunogenic peptides to relevant HLA molecules suggest that there may be differences as well between peptides that induce disease in Mestizo and Asian populations. Purpose: To evaluate Killer Immunoglobulin-like receptor (KIR) genes in patients with uveitis. Methods: Individuals diagnosed with birdshot chorioretinopathy (BCR) (n = 19) and Vogt-Koyanagi-Harada (VKH) disease (n = 25) were evaluated for both activating and inhibitory KIR genes as well as Class I human leukocyte antigen (HLA) specificities using polymerase chain reaction protocols. The presence or absence of the appropriate HLA ligands for inhibitory genes was established. The individual genotypes, the patterns of gene inheritance, and the ratio of activating:inhibitory genes with their HLA ligands were compared to local and published Caucasian (BCR) or Mestizo (VKH) controls. Results: The ratio of activating:inhibitory KIR genes with their HLA ligands was elevated compared to controls both forms of uveitis. In addition, patterns of activating genotype not found in any controls were found. Conclusion: The ratio of activating:inhibitory KIR genes is increased in individuals with BCR and VKH disease. The fact that a similar pattern was found in two different forms of uveitis with distinct clinical syndromes and known HLA associations implies that increased activating:inhibitory KIR genes may be a marker of risk for uveitis. That a system in monitoring 14 infectious diseases with outbreak potential is essential for its containment, thus control if not eradication. Lack of Association between Interferon-Gamma Aim: Determination of susceptibility to tuberculosis with polymorphism of IFN-gR1 gene.Material and Method: Study was prospective case-control. Fifthy patients with smear & Culture positive tuberculosis have been chosen randomly. They were matched with 54 healthy controls with no history of TB.Polymorphism at 395 codon of IFN-gR1 gene was detected with Newport method. Data were analyzed with SPSS version 11.Results: Mean age of patients and control were 55 F 20 and 53 F 13.5 years respectively. Demographic characteristic had no difference within two groups. (P-value N.05) one patient in case group had heterozygote mutation at IFN-gR1 gene. In control group there were no mutations.Conclusion: Genetically susceptibility to TB is not in 395 codon of IFN-gR1 in Iranian TB sample and polymorphism of this loci has occured in 2% of TB patients and 0.96% of total study population. 1 1 Clinical of Immunology, Rostov State Medical University, Rostov-on Don, Russian Federation.Background. Polyclonal activation of B-lymphocytes is one of the basic mechanisms of the HIV pathogenesis, however, the resulting anti-HIV antibodies show no protective effects. In this connection, it is expedient to study the functional properties of antibodies, in particular, their affinity.Methods. We observed 88 patients aged 25-45 and infected with HIV-1, of which 42 patients were at the stage of generalized lymphoadenopathy (LAP), 34 persons at the stage of pre-AIDS, 12-at the stage of AIDS. ELISA was used to determine the anti-HIV antibodies titer, the degree of their affinity (AK).Results. It was found that at the LAP stage the content of immunoglobulins at this stage of disease was IgA-1,59 F 0,42g/l; IgM-1, 02 F 0,20g/l; IgG-10, 90 F 1,26g/l, but the anti-HIV antibody titer was lg 4,30 F 0,14, and the degree of their affinity (AK) was 30 F 6 units. Thus, the development of the infection process is characterized by aggravation of the insufficiency of the functional activity of the anti-HIV specific antibodies, confirmed by the marked reduction of their affinity. Object: Virus C hepatitis (HCV) often has a more favorable course in younger patients. Considering the involution of the thymic function with age, we investigated the output of recent thymic emigrants in HCV patients.Matherials and methods: To evaluate recent thymic emigrants, we used a competitive quantitative PCR in order to determine the percentages of cells with Cj-T cell receptor excission circles (TREC). This study was performed in 13 HCV patients at diagnosis and before any anti-HCV treatment. The results obtained in this group were compared to those obtained in a gruop of 17 agematched controls.Results: We found that in the 13 HCV patients naive for anti-HCV treatment percentage of TREC was 3%. We could not detect a correlation between the percentages of TREC and the patientsT viremia. In contrast, in the 17 age-matched controls the percentage of TREC detected by us was 6% (P = 0.02).Conclusions: Our study describes a novel immune defect in HCV patients. Additional studies are needed to get further insight in the possible role of TREC defect in the pathogenesis and prognosis of the disease. Background: IgE antibodies play a major role in the pathogenesis of typ I allergies. As the half life of serum IgE is short, plasma cells continuously have to secrete large amounts of IgE to maintain the serum titers over long periods of time. It is currently debated, whether IgE-secreting plasma cells are short-lived end products of a chronic activation of B cells, or long-lived, if maintained in supportive niches of the bone marrow or in inflamed tissue. Method: We have analyzed proliferation and lifetime of IgE-secreting plasma cells in an ovalbumin (OVA)-specific, murine allergy model. The time point of origin and the plasma cell turnover in the spleens, lungs, lymph nodes and the bone marrow of OVA allergic mice were determined according to incorporation of BrdU into DNA of proliferating cells. Organs and sera were analysed using ELISPOT, ELISA, fluorescence microscopy and flow cytometry. Results: 4-6 weeks old mice were sensitized with OVA and then continuously fed BrdU for 2 weeks, supplied via their drinking water. 25% of IgE-secreting plasma cells in spleens of the OVA allergic mice were BrdU-positive, indicating that they had proliferated within the time of BrdU-feeding. 75% of the IgEsecreting splenic plasma cells had been generated before that time period and thus had a lifetime of more than 2 weeks. Antiproliferative, immunosuppressive therapy (cyclophosphamid) did not eliminate the cells producing OVA-specific IgE antibodies, indicating that the respective plasma cells are not dividing and long-lived. Conclusion: IgE-secreting plasma cells can be longlived. These long-lived, IgE-secreting plasma cells provide allergen-specific IgE independent of the presence of allergen and are resistant to immunosuppression. Transmission Hepatic Electron Microscopic Findings in Chronic Experimental Schistosomiasis Mansoni after Praziquantel and an Antifibrotic. This study aims to evaluate the effect of a drug combination: praziquantel (CAS 55268-74-1 EMBAY 8440, Biltricide), and an antifibrotic agent (B _ Amino Propionitrile Mono fumarate salt (2079-89-2), upon the transmission hepatic Electron microscopic findings in chronic experimental murine schistosomiasis mansoni. This group was further subdivided into four small subgroups. Sacrifice was done 13 weeks later, a time needed for the infection to develop into a chronic one. Sacrifice was done 5 weeks later. Subgroup III: infected mice given B-Amino Propionitrile daily as 5mg powder in 0.5ml saline for 14 successive days. These changes were less evident in the other previously mentionned groups.Key words: Chronic Schistosomiasis mansoni, Primary infection, challenge or secondary infection, Beta amino propionitrile, Praziquantel., Transmission Electron Microscopy. Transmission Hepatic Electron Microscopic Findings in Acute Experimental Schistosomiasis Mansoni after Praziquantel and an Antifibrotic. This study aims to explore the repercussions of a drug combination: praziquantel (CAS 55268-74-1 EMBAY 8440, Biltricide), and an antifibrotic agent (B _ Amino Propionitrile Mono fumarate salt (2079-89-2), upon the transmission hepatic Electron microscopic findings in acute experimental murine schistosomiasis mansoni.In this study, a group of 100 Swiss albino mice was used. This group was further subdivided into four small subgroups. Subgroup III: infected mice given B-Amino Propionitrile daily as 5mg powder in 0.5ml saline for 14 successive days. Agaim, there was resorption of the previously deposited collagen fibres in the intercellular matrix. These findings are the main stigmata of hepatic cellular regeneration.These data were less salient in mice given Praziquantel or Beta aminopropionitrile alone. It is also a trial to elucidate the repercussions of this drug combination upon worm load and percent resistance to reinfection. Moreover, it aims to study liver enzymes level (Alanine aminotransferase and Aspartate amino transferase AlT & AST) in experimental murine schistosomiasis mansoni.In this study, a group of 100 Swiss albino mice was used. This group was further subdivided into six small subgroups. Subgroup V: infected mice given B-Amino Propionitrile daily as 5mg powder in 0.5ml saline for 14 successive days. Sacrifice was done 112-124 days post primary infection.Mice given the combination regimen Praziquantel + Beta Aminopropionitrile (PZQ + BAPN), compared to those given each drug solely, revealed absence of worm recovery at perfusion, the highest score of percent resistance to reinfection, and a 19.6+0.9 & 18.3+0.9 IU/L serum alanine aminotransferase & aspartate aminotransferase levels respectively. These data were less salient in mice given Praziquantel or Beta aminopropionitrile alone.Key words: Chronic Schistosomiasis mansoni, primary infection, challenge or secondary infection, Beta amino propionitrile, Praziquantel. Serum transaminase levels (ALT &AST). The goal of this study, is to evaluate the effect of a combination between an anthelmintic drug praziquantel (CAS 55268-74-1 EMBAY 8440, Biltricide), and a muramyl dipeptide derivative Adamantylamide Dipeptide (AdDP) {CAS 768-94-5 (Amantadine)} in potentially tolerized Schistosoma mansoni infected, egg-injected C57BL/6 mice. It is also a trial to elucidate the repercussions of this drug combination upon worm and tissue egg loads and oogram pattern. A group of 120 C57BL/6 mice was used in the experiment. This group was further subdivided into five small subgroups. Effect of a Novel Muramyl Dipeptide Derivative Egg-injected mice given the combination regimen Praziquantel + Adamantylamide Dipeptide (PZQ + AdDP), compared to infected untreated control, revealed absence of worm recovery at perfusion and 100 % dead ova in the oogram. Again an evident reduction in the hepatic and intestinal tissue egg loads was recorded in this group compared to infected untreated (wether egg injected or not) control mice. Large amount of CpG-S ODN as well as bacterial DNA could trigger over inflammatory response, even sepsis. Therefore, it is important to balance the activity of CpG-S ODN and reduce the release of cytokines induced by CpG-S ODN. Despite comparable levels of unmethylated CpG dinucleotides, DNA from serotype 12 adenovirus (Adv12 DNA) is immunestimulatory, but DNA from serotype 2 and 5 (Adv2 DNA, Adv5 DNA) are nonstimulatory and can even inhibit activation by bacterial DNA. Based on the above difference, Krieg found some neutralizing CpG ODN (CpG-N ODNs). However, Zhao found above CpG-N ODN had no inhibitory effects even when the concentration ration reached to 10:1. Therefore, it seems the CpG-N ODNTs sequences should be investigated further. In the present experiments, we searched for CpG-N ODN in Adv2 and Adv5 DNA, DNA after comparing the sequence differences between Adv2 DNA, Adv5 DNA and Adv12 DNA, Escherichia coli DNA (EC DNA). Nineteen specific CpG motifs and 12-nucleotide sequences in Adv2, 5 DNA were ascertained after numbers and frequencies of 256 kinds of CpG motifs in Adv2, Adv5, Adv12 or EC DNA were calculated. However, none had been found to have the properties of CpG-N ODN after their assays on TNF-a release induced by CpG-S ODN. Accidentally, we found there existed a relationship between free energy and bioactivity. Therefore, putative CpG-N ODN were re-discovered and investigated. In our in vitro experiments, CpG ODN208, without cellular toxicity, inhibited TNF-a release from hPBMC or RAW264.7 induced by CpG-S ODN in a dose-and time-dependent manner. In our in vivo experiments, CpG-N ODN208 could markedly protect mice from lethal challenge by CpG-S ODN and significantly decreased TNF-a release in mice. Above results suggested that there existed a relationship between free energy and bioactivity of CpG-N ODN, and strong inhibitory CpG-N ODN could be screened based on this kind of relationship. Background: Visceral leishmaniasis is a fatal disease, which is caused by Leishmnia spp. After inoculation of promastigotes by sand fly, some individuals can develop type-1 immune response and control the infection, while others may be involved with acute form of the disease. It seems that IL-10 can inhibit type-1 immune response and cause more severe form of the disease. The aim of the this study was to evaluate the IL-10 serum levels in the different courses of the disease.Methods: Thirty two patients with visceral leishmaniasis were included in this study. Blood samples were collected from the patients in three phases: at the time of diagnosis, after medical therapy (when the patients became afebrile), and at the time of discharge. Sera were separated and stored at -708C until cytokine assay. IL-10 level was determined using ELISA method.Results: The mean IL-10 serum levels were significantly different in three phases of sampling (P = 0.002). The mean F SE of IL-10 serum levels in three phases were 77.4 F 15.6 pg/ml, 55.8 F 18.6 pg/ml and 17.7 F 4.9 pg/ml, respectively.Conclusion: According to our results higher levels of IL-10 at the time of diagnosis may act as an inhibitory factor for cellular immunity and have a role in the development of visceral leishmaniasis. It seems that after medical therapy, IL-10 serum level decreases significantly, which may be associated with recovery.Sa2.12. IL-10 Gene Polymorphisms and Susceptibility to Brucellosis. Background: Brucella spp. Is a gram-negative facultative intracellular bacterium and causative agent of brucellosis. It is clarified that type-1 immunity is important to control Brucella infection. In this regard, macrophages have critical role. IL-10 is a Th2-type cytokine that inhibit macrophage activation. It is known that production of IL-10 is affected by its gene promoter polymorphisms. In this study we investigated the relationship between IL-10 gene promoter polymorphisms and susceptibility to brucellosis.Methods: One hundred and ninety patients with brucellosis, 186 healthy individuals who were members of patientsT family and 82 healthy animal husbandmen who had infected animals with Brucella were included in this study. All individuals were genotyped for three bi-allelic IL-10 gene promoter polymorphisms at positions -1082(G/ A), -819(T/C), and -592(A/C) using PCR-RFLP.Results: Genotype and allele frequencies of -592(A/C) and -819(T/C) were significantly different between patients and animal husbandmen groups (P b 0.05).Conclusion: There are some reports showed that A allele at position -592 of IL-10 gene is associated with lower IL-10 production in-vitro or in-vivo. According to the results, higher frequency of A allele at position -592 in animal husbandmen may cause these individuals more resistant to disease. Background: Although correlation between kala-azar disease and several factors have been determined, some unknown factors also exist that need to be recognized. Protective immunologic response against leishmaniasis are characterized by a strong cellmediated immune response. Since cytokine gene polymorphisms may associated with different ability for cytokine production, the aim of this study was to investigate the relationship between IFN-g gene polymorphism and kala-azar.Methods: We genotyped 122 patients with kala-azar, 63 patientsT siblings who were healthy, and 103 healthy individuals who were resident in endemic area and had positive Leishmanin skin test. Genomic DNA was extracted from blood samples and IFN-g gene polymorphism at position +874 (T/A) was determined by allele specific polymerase chain reaction (ASPCR) method.Results: The frequency of TT genotype in patients was significantly less than their siblings (22.1% and 30% respectively) [P = 0.021], while no significant difference was detected between patients and healthy individuals resident in endemic area (P = 0.35).Discussion: Cell-mediated immune response is an effective immunity against Leishmania and IFN-g play a fundamental role in cellular immunity induction. Some researchers reported the association of IFN-g +874 TT genotype with higher IFN-g production. Therefore, it seems that higher production of IFN-g in patientsT siblings may help them to be more resistant to infection.Sa2.14. Polymorphisms of IL-10 Gene Promoter in Patients with Kala-azar. Background: Visceral leishmaniasis is caused mostly by Leishmania infantum in south of Iran. Manifestations range from asymptomatic infection to fatal disseminated visceral disease. Protective immune response against Leishmania is cell-mediated immunity and it is known that IL-10 can down-regulate this kind of response. Researchers showed that polymorphisms in IL-10 gene promoter can regulate IL-10 production. The aim of this study was to determine the relationship between IL-10 gene polymorphisms and outcome of the disease.Methods: One hundred and twenty pediatric patients involved with kala-azar, 57 healthy individuals who were patientsT siblings and 102 healthy individual who lived in endemic area without any history of kala-azar or cutaneous leishmaniasis and with positive Leishmanin skin test were included in this study. Polymorphisms of IL-10 gene promoter (-1082G/A, -819T/C, -592A/C) were determined using PCR-RFLP.Results: There were no significant differences in genotype and allele frequencies of investigated IL-10 gene polymorphisms between the groups.Conclusion: It is documented that protective immunity against leishmaniasis is cell-mediated immunity. Therefore the presence of Th2-type cytokines during the disease can worsen the condition of the patients. Since the results showed no significant differences in genotype and allele distributions between the groups, study of the cytokine profiles and other cytokine gene polymorphisms are recommended. During infection by Trypanosoma cruzi, the etiological agent of Chagas disease, both cellular and humoral immune responses are essential in controlling the parasitemia. While this immune response is necessary for protection it is also responsible for the morbidity caused by the infection. CD25+CD4+ regulatory cells (Tregs) represent a unique lineage of T cells that have an important role in controlling immune responses against self and foreign antigens. We wanted to investigate if Tregs played any role in controlling immune responses during T.cruzi infection. C57BL/6 mice were depleted of CD25+ cells (injected with monoclonal antibody PC61) or were injected with an isotype matched control (GL113) and infected with parasites of the strain Colombiana (strain that elicits an intense myocarditis). Both groups were evaluated for the amount of circulating parasites, mortality rate, immunological parameters and histological analysis of the heart throughout the course of the infection. Animals depleted of CD25+ cells had decreased levels of parasites in the bloodstream and decreased mortality rate. This is associated with an augmentation of activated T cells and better production of pro-inflammatory cytokines. CD25+ depleted animals also display a more severe inflammation of the cardiac tissue when compared to control animals.Sa2.16. Dendritic Cell Mediated Immune Response Is Impaired by the Mycobacterium tuberculosis Mannosylated-LipoArabinoMannan. ManLAM may have a counteractive effect on DCs towards a fully protective inflammatory response. Here, we investigated the impact of ManLAM or PiLAM (LAM from M. smegmatis capped with phosphoinositide residues) on DC maturation and function using combined approaches of phenotyping, cytokines release, NK cell activity and T-cell priming. In contact with ManLAM, DCs displayed a pattern of partial maturation including the intermediate expression of MHC class I and class II molecules and a low expression of the costimulatory molecules CD80 and CD86 compared to fully LPS-matured DCs. They cannot be considered as immature DCs because they lost their FITC-dextran phagocytic activity. At the opposite, they do not present a fully matured phenotype. Indeed, ManLAM-incubated DCs are still sensitive to autologous NK lysis and are not able to prime naive T-cell responses. Absence of NK lysis was noticed when ManLAM-DCs were incubated with CD40 antigen, confirming a partial maturation of DCs with ManLAM only and the need of a second signal to complete maturation. Altogether, the overall effect could be an impaired innate immune response towards M. tuberculosis. Inflammatory Response and Apoptosis of Polymorphonuclear Neutrophils by Prevalent Strains of Mycobacterium tuberculosis. 1 1 Department of Immunology, Tuberculosis Research Centre, Chennai, Tamil Nadu, India.Background: Macrophages and Polymorphonuclear Neutrophils (PMN) are the professional phagocytes involved in antibacterial defense. The PMN influx, the first line of defense, occurs as the early response to curtail the mycobacterial infection. Mycobacterium tuberculosis (M.tb) induced activation leads to proinflammatory response and apoptosis of PMN. Objective: We characterised two prevalent strains of Mycobacteria (S7 and S10) showing differential immune response in PPD positive population. Here, we aimed to study the efficacy of these strains to induce apoptosis and modulate the expression of surface molecules and cytokine secretion in PMN of TB patients. Methods: PMN were isolated from RBC pellet obtained from Ficol-Hypaque gradient centrifugation and further subjected to sedimentation in 3% Dextran. PMN were infected with various mycobacterial strains (S7, S10, and H37Rv) at Multiplicity Of Infection (MOI) of 3:1 and incubated for 3 and 18hrs. The Phagocytic index, percentage of apoptotic Neutrophils (Annexin V-FITC positive by FACS), cell phenotypes (CD16 and CD69 by FACS) and cytokines (TNF-a and IL-1b by ELISA) were assessed. Results: A significant increase in Annexin V positive cells with corresponding decrease in CD16 and CD69 expression was observed with S7 and S10 strains when compared to uninfected control after 3hrs of infection. Further decrease in CD16 expression was observed at 18hrs but no significant change in Annexin V positivity. Conclusions: Clinical strains down-regulated CD16 expression and inhibited de novo synthesis of an early activation marker, CD69 on TB-PMN. These strains also showed a significant increase in apoptosis after infection thereby reducing the number of phagocytes and escaping from the intracellular lytic microenvironment. Thus clinical isolates were able to inhibit the early activation of Neutrophils and thin out the killing mechanisms for their own survival.Sa2.18. Comparison of Regional and Systemic Humoral Immune Response to a Parasitic Infection. The kinetics of humoral immune response against Trichinella spiralis (TS) were characterized with immunofluorescence assay. The mesenteric lymph nodes (MLN) and the spleen of infected rats were examined for concurrent expression of multiple antibody (Ab) isotypes from day 1 to day 15 after infection. The tissues were processed and stained with either a pan-B cell marker (OX33) conjugated with rhodamine (XRITC) or combinations of dual monoclonal Ab probes plus secondary Ab conjugated with XRITC or fluorescein (FITC). As compared to the uninfected controls, the MLN and the spleen showed significant proliferation of dual-Ab expressing B cells (Debc) on days 7 and 10 respectively, with the regional immune response proceeding ahead of the systemic response. During the immune response, only minimal numbers of B cells expressed single Ab isotype while most B cells expressed more than one isotypes of Ab. When combining all the numbers of Debc within each tissue for each of the respective days, and comparing those numbers with the total numbers of B cells that were OX33+ in the serial sections of the same tissue specimens, the combined Debc in the spleen were N6 times higher than OX33 labeled B cells on day 10, and the Debc in MLN were N 3 times higher than OX33+ B cells on day 10. Our results thus indicate that the Debc were most likely expressing more than two isotypes on the surface during the peak days of the humoral immune response to the pathogen and such phenomenon occurred in both immunologic tissues. Co-Administration of Corticosteroids (CS) with Anti-Tuberculous Drugs (ATD) in Tuberculous Menangitis Had Not Only Reduced the Intra-Cranial Pressure Symptoms, Also Had Increased PatientTs Compliance.M. Ishaq, I. M. Sameera. 1 Allergy/Pulmonology, Al-Junaid Hospital, Nowshera, Pakistan.Purpose: Patients with tuberculous meningitis, under ATD regimen alone though had been associated with an improvement in the clinical manifestations, but still there been sporadic incidences of residual manifestations on completion of ATD, concerning cerebral edema from chronic inflammatory changes of tuberculoisis.Cncomitant administration of (CS) had significantly improved the clinical outlook.Methods: Patients with tuberculous meningitis, complaining of evening rise of temperature & manifestations from raised intracranial pressure i.e, confusion,anorexia,diffuse head ache, irritability lethargy, on ophthalmoscope there had edema of the optic disc, then one group of(n = 8) patients had been medicated with (ATD) alone(ATD-) another group of (n = 10) patients with (ATD) & (CS)(ATD+). For adultTs prednisolone 40mg/day in divided dose for 7-10days followed by 20mg/day in divided dose, then tapering the dose according to the therapeutic response by 5 mg weekly for 8-9weeks.For children prednisolone 1-2mg/kg body weight for 7-9weeks. In another trials Dexamethasone in dose of 0.15mg/kg body weight four times/day for 1-2weeks, then discontinued in a tapering fashion over 4weeks had been also found beneficial. On completion of the treatment, Patients with (ATD+) had a uniform therapeutic response with almost insignificant incidence of residual symptoms, where as Patients with (ATD-) alone comparatively been associated with occasional head ache, altered sensorium etc.Results: (CS) in the therapeutic trials had the beneficial role of resolving portentous CSF resulting from inflammatory changes. also had improved patients compliance concerning intake of (ATD).Conclusions: From the later on follow up of patients, there was no incidence of relapse in either group, but (n = 2) patients treated with (ATD-) still had been medication for head ache & convulsion etc.Clinical Implications: Patients with (ATD+) had been provided with steroid medication card on completion of treatment. Methods The persons including of following six groups have been studyed. Group B, 3 cases of the doctors or nurses who were infected by SARS in the working despite with careful prevention. Group C,8 cases of pneumonia who was treated in hospital with SARS patients but no SARS; Group D, 22 intensive SARS patients; Group E, 36 mild SARS patients and Group F, 21 cases of healthy children who have no antibody responses with HBsAg injection. The machine of Array 360 has been used to detect the gross of IgM, IgG and IgA in plasma. The lymphocytes subtype of CD3, CD4, CD8 and CD56 have been assayed by flow cytometer. The number of CR1 on erythrocyte was detected by cell-ELISA. PCR and RFLP have been used to analysis the genomic density polymorphism of ECR1. The immune reactivity of lymphocyte to foreign antigen will be observed by dynamic microscope. Results: The gross of IgM, IgA and IgG Group A, C and F are all in lower level than that of normal individuals, especially in IgM (P = 0.0014), and the reactivity of their lymphocyte to antigen is also in lower levels. The percent of NK cells is significantly higher than that of normal. By contrary, the levels of IgM in group B, D and E are significantly higher than normal individuals, and their lymphocytes are easily destroyed by antigen stimuli. The numbers of CD3/CD4/CD8 are all in low levels. The number of ECR1 in intensive SARS patients are significantly lower than that in mild SARS patients but the former CR1 are most in HH genotypes. Conclusion: There is significant relationship between the higher immune responses and the SARS infection. The immune destroy may be the main pathogenesis of SARS. A. U. Bielinska, K. W. Janczak, J. J. Landers, J. R. Baker, Jr. 1 Internal Medicine and Center for Biologic Nanotechnology, University of Michigan, Ann Arbor, MI, USA.Rationale: Current, live virus vaccines for smallpox have unacceptable side effects. The nanoemuslion works by first removing viral envelope proteins then triggering phagocytosis of the proteins into antigen presenting cells in the mucosa.Methods: A NE approved for human use was mixed with VV (Western Reserve serotype). Mixtures of NE and VV were applied to the nares of mice. Systemic and mucosal antibody and cellular immune responses were then evaluated, and animal sera were tested for the ability to neutralize virus.Results: A brief incubation with 10% nanoemulsion led to greater than a six-log reduction of virus titer. Anti-VV mucosal IgA and serum IgG were detected three weeks after a single intranasal administration of NE/vaccinia mixture, and the immune response was optimized in animals vaccinated 2-3 times. High titers of VV neutralizing antibodies were detected in mice immunized with NE-killed virus. Virus-specific Th1 immunity (INFg production) was observed in splenocytes from immunized animals. No evidence of protective immunity was observed in control animals immunized with formalin-killed virus. No animal had evidence of viral replication after vaccination, documenting the complete inactivation of the virus by NE.Conclusions: VV inactivated by NE is immunogenic and can serve as a killed virus mucosal vaccine for small pox. The presence of NE is necessary for the development of robust protective mucosal and systemic immunity. Background In the immunocompromised host adenovirus can cause fatal infections, especially after stem cell transplantation. No specific antiviral therapy of proven value currently exists for severe adenoviral infection. A potential form of treatment is adoptive therapy infusing adenovirus specific T-cells from the donor. The aim of the study was to identify target epitopes of adenovirus and to capture and characterize adenovirus specific T-cells. Novel Pan-DR Binding T-Cell Epitopes of Methods Eleven proteins of adenovirus type 5 were selected. By using a computer algorithm designed to predict HLA pan-DR binding Tcell epitopes, we selected 19 peptides based on predicted high affinity to multiple HLA-DR alleles and a high degree of homology with other adenovirus serotypes. Peripheral blood mononuclear cells (PBMCs) from 26 healthy adults were isolated and incubated with these peptides. Proliferation expressed as Stimulation Index (SI) was determined. Six peptides with highest SI were selected. In ten subjects the cytokine and chemokine profile induced by these epitopes was determined with Multiplex Immuno-assay (MIA). In addition PBMCs were cultured with complete inactivated adenovirus and restimulated with the different peptides. Subsequently, with MIA cytokine production was measured. Moreover, with the use of T-cell capture method 1 and FACS-sorting it was possible to induce and capture adenovirus specific T-cells. With polymerase chain reaction (PCR) characterization of adenovirus specific T-cells was performed. Results Six pan-DR binding epitopes of adenovirus were selected based on a positive proliferation response in a large percentage of donors, namely E1B protein (65% of donors), 2 peptides of hexon protein (58% and 73%), DNA-polymerase (57%), E3A-glycoprotein (46%) and fiber protein (34%). These epitopes induced a predominant proinflammatory cytokine and chemokine profile. Also after culturing with complete inactivated adenovirus and restimulation with the adenoviral peptides, cytokine profile showed a pro-inflammatory pattern, suggesting that peptides are naturally processed. By using T-cell capture method adenoviral peptide specific CD4+ T-cells were identified and sorted. Preliminary data show that such adenovirus specific T-cells display a high expression of TGF-1 beta, IFN gamma and Tbet (Th1 response), but remarkably also expression of GATA3 and IL10 (Th2 response).Conclusion With a pan-DR binding computer algorithm it was possible to identify HLA-class II restricted T-cell epitopes of adenovirus type 5. These peptides induce a predominant pro-inflammatory cytokine profile and also seem to be naturally processed. With T-cell capture method it was possible to capture and characterize the adenovirus specific T-cells. This is an important step towards adoptive immunotherapy by infusion of adenovirus specific T-cells. 1 Inflammation usually occurs in extravascular tissues following the invasion of micro-organisms, such as bacteria, to the body. These sites also contain immuno-modulators (e.g., chemokines, cytokines, acute phase proteins), and leukocyte-derived extracellular matrix-specific enzymes, such as heparanase and elastase, which can modify the composition of the tissue, and probably also degrade certain inflammatory mediators, thus yielding putative bioactive products. We postulate that these new small molecular weight mediators can exert effector functions needed to evoke or terminate inflammation.Herein, we identified novel immunoregulatory compounds in the degraded products of human body fluids, especially in wound fluids of chronic leg ulcers of diabetic patients. Thus far we were able to isolate and identify several amino acid sequences and synthesized, and examined them in vitro and in vivo. Among these peptides, two are derived from apolipoprotein A-1 and two from fibrinogen. Treatment of purified human T cells with these peptides down-regulated nuclear factor-nB activity and reduced the secretion of TNF-a and interferon-g. We suggested that these peptides may terminate inflammatory reactions by transmitting negative signals to the inflammationinducing leukocytes.Our results indicate that by using this approach we may have found a first set of such novel anti-inflammatory peptides. We hope that such peptides can be used to down-regulate inflammatory reactions in vivo in human patients suffering from chronic inflammatory diseases. The Inflammatory & Immunological Complex Clinical Presentation of Mycoplasmal Infection in School Children.M. Ishaq, I. M. Sameera. 1 Allergy/Pulmonology, Al-Junaid Hospital, Nowshera, Pakistan.Purpose: Outbreaks of mycoplasma infection had been associated in school children/camps at early and late winter seasons, sometimes with a considerable number of hospital admissions & loss of school days amongst children & adolescents.Methods: In an under developed community with bunch of schools, children& adolescents age 5-16 years mean age10 F .5 years. Presenting features i.e,raised temperature 95% cases, malaise 85-90%, cough 80-85, headache 80%, medium to fine rales 60-65%. On Laboratory investigation were leucocytosis (10,000-20,000/m 3 ) 20-30% cases, raised ESR, variable Pulmonary segmental/lobar consolidations seen, cold agglutinin level 1:4-1:256 titer appears in blood of 50% of children, most of the cases had raised indirect of fluorescent antibody in the range of 1:10-1:320. M. Pneumoniae.Results: Ignored/late treated cases from the distant locations, almost 0.1-0.9% had transverse myelitis, encephalitis menangoencephalitis proved by CSF, PCR, culture studies. myopericarditis, haemopericardium heart block, 0.02-0.1% etc; 2-4% with mucocutaneous manifestations,i.e, erythematous maculopappuler erruptions, steven jhonsonTs syndrome, 4-5% with joint manifestation i.e. 10-20% of complicated cases had nausea, vomiting, altered bowel habits, etc; Conclusions: Mycoplasma infection is a contagious disorder resulting from substandard hygienic living, poor school health (failure of provision of well ventilated classrooms/Isolation of infected cases) etc; Clinical Implications: Mycoplasma pneumonia is held responsible for 20% cases of community acquired pneumonia. Objective: To establish a experimental system of war against yeast cells in hemaimmune reaction road map.Methods: 0.2ml suspension of yeast cells (5Í10 8 /ml) (or 0.2ml NS as control) were added into 0.2ml fresh anticoagulant (citric acid) whole blood or 0.2ml white blood cells, added 0.3ml plasma (or 0.3ml NS as control), and incubated for 1h at 378. Further more, we added 0.2 ml red blood cellls into the tube abovementioned respectively, and incubated for 1h at 378. The hemaimmune reactionary activity was assessed by checking blood cell adhering rate to yeast cells, CD35, DARC (Fy6), CXCR4, IL-8, IL-6, CD25, gene activation (Fy6 gene) et al.Results: Yeast cells can activate hemammune reaction road map experimental system showing change of various indexes. Yeast cell activation method will be a useful method to study the relationship between whole blood cells and plasma and the relationship between red blood cells and white blood cells in hemaimmune reaction.Conclusion: It can provide a useful method for innate and adaptive immunity study and for immune regulation study and for the theory study of hemaimmune reaction road map in clinic. Phage Display Epitope Study of Two Toxins Produced by Enteroaggregative Esherichia coli That Cause Pediatric Diarrhea: Identification and Characterization of Antigenic and Immunogenic Mimotopes. In earlier publications from this laboratory, two toxins a of 104kDa (Pet) and other of 114 kDa (Pic), members of the SPATES (serine-protease autotransporters from enterobacteriaceae) family were described as molecular agents associated with pediatric diarrhea caused by Enteroaggregative E.coli, especially in developing countries. Despite different location of their genes, in the chromosome (Pic) and in a plasmid (Pet), the proteins showed a high level of sequence identity. Both proteins have been identified in the same patient and are immunogenic, however, neither their epitopes nor immunological relationships with each other and with the disease are known. Collections of peptides with properties of immunodominant epitopes that elicit the immune responses in patients, would be very beneficial for the research advancing into the immunology of the proteins and their relationships with the disease. To the identification of such peptides we screened gp3 random phage-display linear and constrained libraries with sera of patients and with rabbit antisera obtained against Pet and Pic proteins. The screening of two highly complex phage libraries resulted in collections of peptides sharing well-defined consensus motifs. Although the motifs did not show similarity to the proteins, the carrying those peptides phage were reactive with sera and induced in mice and rabbits antibodies reactive against Pet and Pic in ELISA and in denaturing Western blot. The results indicate that they mimic epitopes containing immunogenic determinants that do not loss their antigenic activity upon denaturation. In addition, the anti-sera against Pet-related peptides showed a cross-reaction with the Pic, and vice versa, i.e. We conclude that the peptides from phage libraries specific to epitopes of these enterotoxins allow further elucidation of their roles in the pathogenesis of diarrhea and are useful in laboratory diagnosis of patient sera. In the ongoing experiments the peptides with best immunogenic and antigenic potentials are used in serological studies of patients and for their ability of inducing neutralization-effective responses in animal models. Chlamydia pneumoniae (Cpn), an obligate intracellular pathogen, is a common cause of respiratory tract infections worldwide and has been considered among other risk factors involved in coronary artery disease. Cpn-infected cells have been reported to be resistant to apoptosis, but it is not clear what component of Cpn is responsible for this action. The present study investigated the role of both nicotine and Chlamydia heat shock protein 60 (cHsp-60) individually and in combination on viability, proliferation and apoptosis in human epithelial cell line (HEp-2). The results of these studies showed that treatment of HEp-2 cells with nicotine significantly increased cell count and viability compared to the untreated controls. Treatment of HEp-2 cells with cHsp-60 did not result in significant changes in cell count and viability. Interestingly, when apoptosis was induced in HEp-2 cells with TNF-alpha, cHsp-60 did in fact significantly increase cell count and viability. In order to ascertain whether this increase reflected a protection against apoptosis, caspase activity was assessed. Results showed that active caspases were down-regulated in cells treated with either nicotine or with cHsp-60. Combined treatment with both nicotine and cHsp-60 resulted in even further down-regulation of active caspases. The anti-apoptotic action of nicotine was blocked by D-tubocurarine chloride, a nicotinic receptor antagonist. The high prevalence of Cpn infection and the ready availability of nicotine in the population lead to concerns about possible combined exposure to both agents. The impact of the combined exposure to nicotine and cHsp-60 on parameters of immune status, in both normal and immunocompromised hosts, warrants further investigation. Twentyeight HIV-1-infected patients, in the stage A of the disease, according to the CDC (Center for Disease Control, Atlanta) criteria, have been studied. We found in 18 out of 28 patients an increase in the number of circulating Vy1 T cells (3-9%) of T lymphocytes (healthy donors: 1-3%). Three of these patients also displayed an increased number of peripheral Vy2 T cells (4-6% vs 2-3% in healthy donors). Moreover, Vy2 T cells were CXCR3 bright and CXCR4 dull , while among the Vy1 subset 50% of the cells were CXCR3-and CXCR4 + . Vy1 and Vy2 T cell clones transmigrate across endothelial monolayers in response to interferon-g inducing protein-10 (IP-10/CXCL10) and stromalderived factor-1 (SDF-1/CXCL12) according to the expression of the specific receptors CXCR3 and CXCR4. In a fraction (10%) of Vy1 T cell clones coexpressing CXCR3 and CXCR4, the homeostatic chemokine 6Ckine/SLC (CCL21) was more effective than IP-10/CXCL10 in driving transendothelial migration of Vy1 CXCR3 + cells, while Vy2 CXCR3 + cells were driven more efficiently by IP-10/CXCL10. IP-10/CXCL10 or 6Ckine/SLC/CCL21 and SDF-1/CXCL12induced transmigration were inhibited by the phosphoinositide-3 kinase (PI-3K) blockers wortmannin and LY294002, supporting that CXCR3 and CXCR4 are coupled to PI-3K. This was further confirmed by the activation of the PI-3K-dependent serine kinase Akt/PKB obtained upon ligation of CXCR3 and CXCR4. In addition, occupancy of CXCR3, but not of CXCR4, led to CAMKII activation; accordingly, the CAMKII inhibitors KN62 and KN93 could decrease IP-10/CXCL10 and 6Ckine/SLC/ CCL21-driven transmigration. Finally, we show that HIV-1 protein Tat, which can be found in the sera of these patients, interferes with the chemotactic activity of IP-10/CXCL10, 6Ckine/SLC/CCL21 and SDF-1/CXCL12 and that the inhibition is due to the cystein-rich domain of the protein, which contains CXC and CXC chemokine-like sequences. This mechanism may contribute to the redistribution of the two gy T cells subset, the resident increasing in peripheral blood in early AIDS and removed from intestine in the advanced disease, supporting the importance of gy T cells in the first defence against HIV-1 infection. Adenovirus infections are usually harmless in healthy children and adults but are serious and potentially fatal in immunocompromised individuals. Like for CMV and EBV-infections, adoptive transfer of specific T-cells is also discussed for adenoviral infections. However, the main adenovirus derived antigenic target of adenovirus-specific T-cell responses has yet to be defined. We here identified the main immunodominant protein of the adenovi-rus-specific CD4 + T-cell response and functionally characterized adenovirus-specific CD4 + T-cell with respect to their cytokine profile. Three major adenoviral proteins including two structural capsid proteins (hexon, penton, polymerase) were recombinantly expressed. CD4 + T-cell-lines specific for the entire set of adenovirus-derived proteins were generated after stimulation of PBMC from healthy adults with adenovirus-lysate 3 (Ad-Lys3) according to antigen-induced CD154 surface expression. Specificity of adenovirus-specific CD4 + T-cell-lines was evaluated after restimulation with Ad-hexon, Ad-penton, Ad-polymerase according to intracellular expression of CD154. Direct qualitative cytokine profile of Ad-specific CD4 + T-cells was assessed after short-term stimulation of whole blood of healthy adults with Ad-hexon and coexpression analysis of CD154 with IL-2, IL-4, IL-10, TNFa and IFNg. Ad-Lys3-specific CD4 + T-cell-lines can easily be generated according to CD154-expression and showed high specificity for the adenovirus derived hexon capsid protein. Only a few Ad-Lys3 specific CD4 + T-cells responded to Ad-penton and none to Adpolymerase. Qualitative assessment of the cytokine profile of Adhexon specific Th-cells proved a Th1-like profile with no IL-4 expressed and dominant expression of TNFa and IFNg. Hexon capsid protein is the main target protein of the adenovirus-specific CD4 + T-cell response in adults that is characterized by a Th1-like cytokine profile. Our results envisage that Adenovirus-derived hexon protein is a candidate antigen for ex vivo generation of adenovirus-specific T-cells in cellular therapies. Objective: SARS is a severe infectious disease caused by a virus identified through gene sequencing and serological analysis as a new strain of human coronavirus. SARS coronovirus (SARS-CoV) causes severe acute respiratory symptoms in patients and results in a high mortality rate. Antigenic peptides recognized by SARS virus specific CTLTs are useful tools for studying the CTL responses during infection enabling researchers to better monitor disease and develop T-cell mediated vaccines. In order to identify potential immunogenic epitopes from the SARS N-protein, we used Beckman CoulterTs iTopia Epitope Discovery System* to analyze the protein across 8 MHC Class I alleles. Identification of SARS T Cell Epitopes Using the iTopiak Epitope Discovery System. Materials and Methods: The iTopia Epitope Discovery System was used to screen the SARS N protein by analyzing all overlapping nonamers (414 peptides) across 8 different HLA-A/B alleles: HLA*A0101, HLA*A0201, HLA*A0301, HLA*A1101, HLA*A2402, HLA*B0702, HLA*B0801, HLA*B1501. The peptide binding assay is the first assay in the system and is a rapid screening of all test peptides across all 8 alleles. All identified binders are further characterized with the ED50 Affinity and Off Rate dissociation assays. The previously identified binders are serially diluted and analyzed in the Affinity assay. An ED 50 value is determined and represents the concentration of peptide needed to achieve 50% binding. These same binders are also analyzed in the Off Rate assay. Peptides are incubated for up to 8 hrs and time points are taken at specified intervals. Results are analyzed using a one phase exponential decay curve and are expressed as t 1/2 value in hrs representing the amount of time needed to achieve 50% dissociation of the peptide from the MHC complex. The Off Rate and Affinity values for each test peptide are evaluated using a multiparametric calculation to generate an iScore. This iScore allows peptides to be systematically ranked for subsequent functional studies. These newly identified peptides can be used to manufacture HLA Class I iTAg MHC tetramers which are an important tool to visualize and quantify the precise in vivo SARS CTL response.Results: Identification of SARS candidate epitopes are as follows: 6 epitopes for HLA*A0101, 30 epitopes for HLA*A0201, 18 epitopes for HLA*A0301, 28 epitopes for HLA*A1101, 37 epitopes for HLA*A2402, 18 epitopes for HLA*B0702, 7 epitopes for HLA*B0801 and 27 epitopes for HLA*B1501. Based on iTopia rankings of candidate epitopes, SARS iTAgk MHC Tetramers were manufactured and used to stain cryopreserved PBMCTs from SARS infected donors.Conclusion: Using the iTopia Epitope Discovery System, the SARS N protein (422 amino acids) was screened and potential immunogenic epitopes were identified based on experimental binding, affinity and off rate determinations. SARS results demonstrate the capacity of the iTopia system to screen large number of MHC Class I restricted peptides and prioritize the epitopes with greatest potential for producing an immune response. * Objective: To study a family with two twins with suspect symptom and bone marrow aspiration signs of HLH and; negative serology for bacteria, virus or parasites in both twins. These twins presented a different degree of clinical severity (Twin 1 had more severity than Twin 2).Methods: Family study perforin expression was analysed by four-color multiparametric flow cytometry COULTER Ò EPICS Ò XL TM Flow Cytometer of Beckman-Coulter (Coulter Corporation). Perforin expression was studied in CD56+CD3-cells, CD56+CD3+ cells, CD8+ CD3+ cells. Genetic analyses were carried out to identify the putative perforin mutation. Genomic DNA was prepared from PBMC using standard protocols, after informed consent was obtained. Exons 2 and 3 of the perforin gene (prf1) were amplified using polymerase chain reaction in the family and in several controls. Quantification of plasma sIL-2R levels was performed in triplicate using commercially-available ELISA kits (Bender MedSystems, Viena, Austria).Results: The family study showed decreased perforin expression in CD 56+CD3-NK cells, CD8+CD3+ cytotoxic T cells in the father and Twin 1, and a normal expression in the mother and Twin 2 when we compared it with healthy controls of similar age. ), in Twin 1 and father in the codon 91 of the exon 2 (GCG-GTG that changes Ala91 to Val). Both were hetero-zygous for this mutation. In all others samples, including Twin 2, no mutations were detected. Although, we did not sequence the entire prf1 gene, it is unlikely that the Twin 2 have another perforin mutation since their expression was normal by flow cytometry.Both twins had higher levels of sIL-2R than healthy controls of similar age; Twin 1 showing a higher level (18.55 ng/ml) than Twin 2 (14.38 ng/ml). In contrast, sIL-2R levels of both progenitors were normal.While, Leishmania parasites were visualized by electron microscope examination on the liver tissue of both twins. Finally Treatment began with antimonials and both patients recovered completely but more quickly Twin 2.Conclusion: Visceral Leishmaniasis associated to HLH syndrome can cause additional and considerable etiologic diagnosis difficulties. However, our results indicate that could be convenience of analysis of perforin defects and concomitant infections in all types of HLH. Leishmania infection is not only able to trigger haemophagocytic syndrome but perforin defects may worsen eradication of leishmania infection. Fc receptor (FcR)-mediated phagocytosis requires the activation of the Rho GTPases Cdc42 and Rac1. However, how Cdc42 and Rac1 are recruited to the FcR is unknown. Here we show that the Ca 2+ -promoted Ras inactivator (CAPRI), a Ras GTPase-activating protein, functions as an adaptor for Cdc42 and Rac1 during FcR-mediated phagocytosis. CAPRI-deficient macrophages exhibit impaired FcgR-mediated phagocytosis and oxidative burst, as well as a defective activation of Cdc42 and Rac1. CAPRI interacts constitutively with both Cdc42 and Rac1, and translocates to phagocytic cups during FcgR-mediated phagocytosis. Importantly, CAPRI-deficient mice have impaired innate immune response to bacterial infection. These results suggest that CAPRI provides a link between FcgR and Cdc42 and Rac1 and is essential for host innate defense. Case Report: An 8 year old Caucasian girl presented with a five year history of recurrent dermatomal vesicular lesions affecting her face and back. At 2 1/2 years of age, the patient developed primary gingivostomatitis with painful, vesicular lesions over her entire oral mucosal surface. She continued to have mucosal outbreaks every other month until 3 1/2 years of age when she complained of facial irritation, soreness, and fevers with subsequent vesicular lesions originating at the corner of her mouth and following the V3 dermatome. Her lesions never cross the midline and occur several times a year triggered by respiratory infections or stress. She continues to have acute outbreaks despite prophylactic acyclovir dosed at 45 mg/kg/day. These infections resolve after standard courses of oral antibiotics. She has no history of lower respiratory tract, bone, joint, deep tissue, or fungal infections. The family history is remarkable for recurrent viral infections in her father and two siblings. Her physical examination was unremarkable with no evidence of lymphadenopathy, hepatosplenomegaly, or chronic rash. A direct fluorescent antibody test performed during a subsequent outbreak was positive for herpes simplex virus and negative for varicella virus in perilesional skin. Multiple viral cultures of lesions at different times were positive for herpes simplex virus. An evaluation for a possible defect in the innate and cell-mediated immune system revealed decreased natural killer cell lysis, an intracellular cytokine assay (ICC) for interferon-gamma production was negative for varicella, cytomegalovirus, Epstein Barr virus, and influenza, a delayed type hypersensitivity (DTH) skin test was reactive to tetanus and mumps, and a lymphoproliferation assay (LPA) for mitogens and tetanus was normal. DTH skin test reactivity and LPA to candida were nonreactive. Humoral immune function was evaluated and immunoglobulin levels of IgG, IgA, IgM, and IgE were normal and IgG2 was mildly decreased. Diphtheria, Tetanus,and Haemophilus antibody titers were protective. Zero of twelve Pneumococcal antibody levels were N1.0 ug/ml before and after vaccination. HSV IgG titers were positive while varicella virus IgG titers were equivocal. Flow cytometry revealed an elevated number of CD 19+ cells.Discussion: The patient presented with recurrent dermatomal vesicular lesions which were caused by herpes simplex virus. Health care providers who typically associate recurrent dermatomal outbreaks with herpes zoster should consider culturing for HSV or other viral etiologies. Although rare, recurrent dermatomal HSV lesions may be a hallmark of an as yet undefined immunodeficiency that may be elucidated as the field of clinical immunology matures. The innate immune defence encompasses a number of cellular and humoral components. Among the latter the complement system plays important roles, and is also involved in establishing an adequate specific clonal immune response. The recently established mannan-binding lectin (MBL, or mannose-binding lectin) complement pathway relies on the recognition of the microorganisms by their pathogen-associated molecular patterns (PAMPs). The oligomeric structure of MBL provides for 12 or more clustered C-type lectin domains, which allows for high avidity binding to suitably spaced sugar residues. This binding triggers the activation of the MBL-associated serine protease, MASP-2, which in turn activates C4 and C2 to generate the C3 convertase, C4bC2b. The level of MBL in plasma varies from less than 10 ng to 5 Ag/ml and is determined by polymorphisms in exon one and in the promoter region. Numerous studies have found association between MBL deficiency and increased susceptibility to infections. It appears that alone MBL deficiency does not predispose to infections, but only when also other elements of the immune system are suboptimal. Thus, e.g., leukaemia patients in chemotherapy are at high risk of serious infections when MBL deficient (Peterslund et al. Lancet,358, 637-8, 2001 ), MBL deficient colorectal cancer patients have increased risk of postoperative infections (Ytting H et al. Cancer Immunol Immunotherapy, online pub, 2004) . MBL deficiency has also been reported to be associated with autoimmune manifestations, and surprisingly, with increased risk of atherosclerotic diseases. Recombinant MBL is now being produced in a human cell line by NatImmune A/S, Copenhagen by methodologies modified from Vorup-Jensen et al. Immunopharmacology, 1, 677-87, 2001; Jensenius et al. 2003 31, 763-7, 2003 and purified to yield a preparation certified for clinical trials. The rMBL shows physical and biological characteristics similar to those of plasma-derived MBL. Preclinical and phase I trials have been conducted and are currently being evaluated. No safety concerns were observed. The renal epithelium is the initial site of contact between pathogens and their hosts. Through this interaction epithelial cells may have the opportunity to detect and respond to pathogens, which leads to activation of inflammatory responses and recruitment of leukocytes. This initial phase of inflammatory response to infection often involves activation of innate immune system in particular the complement. A series of fluid phase and cell surface inhibitors, including Membrane Cofactor Protein (CD46), exist to prevent excessive complement activation. CD46 is also known to be the receptor for various types of pathogens. Here we have investigated the potential of CD46 to mediate signal transduction and subsequent internalisation in human renal epithelial cells. CD46 was found on both apical and basolateral surfaces of cells by staining with anti-CD46 antibody and a fluorochrome. Introduction of a secondary antibody brought clustering of receptors. Incubating at 37 8C led to internalisation of antibody, which was not seen when incubating at 4 8C. Extracellular antibody was detected by a greenlabelled fluorochrome. Cells were then fixed and permeablised, and a red-labelled fluorochrome was used to detect intracellular and extracellular antibody. However, internalisation was independent of src kinase activity. Here we present data demonstrating that antibody can be internalised specifically through its binding to CD46 receptor, which suggests that CD46 may play an active role in mediating signalling events. It is our interest to investigate whether CD46 can serve as a receptor for mediating internalisation of opsonised pathogens upon infection.Sa2.36. Access to the Entire Human Antigen-Specific CD4+ T-Cell Response According to Antigen-Reactive CD154 Expression.Marco Frentsch, 1 Olga Arbach, 1 Dennis Kirchhoff, 2 Thomas Schneider, 3 Alexander Scheffold, 2 Andreas Thiel. 1 1 Clinical Immunology, German Arthritis Research Centre, Berlin, Berlin, Germany; 2 Immunmodulation, German Arthritis Research Centre, Berlin, Berlin, Germany; 3 Gastroenterology and Infectious Diseases, Charite-Campus Benjamin Franklin, Berlin, Berlin, Germany. Current techniques to assess antigen-specific T-cells suffer from various limitations: Either the access is restricted to activated cells that reacted with the expression and secretion of cytokines or could only be performed with a so far rare selection of specific peptide MHC-II multimers. We report here on a new method to assess the entire fraction of CD4+ T-cells specific for a particular antigen independent of their cytokine memory.For this we have employed here the intracellular and extracellular detection of antigen-induced CD154 expression after in vitro stimulation with various antigens such as CMV, TT, Adenovirus, birch allergen, SEB and Mycobacterium tuberculosis ESAT-6. In the course of antigen-driven in vitro activation of CD4+ T-cells CD154 is specifically expressed by antigen-specific CD4+ T-cells. Antigen-specific CD154 expression is detectable intracellularly in fixed CD4+ T-cells when stimulations are performed in the presence of Brefeldin A facilitating co-expression analysis of cytokines. Moreover, we developed a strategy to circumvent the rapid internalisation of reactive surface CD154 expression after interaction with its counterpart CD40. This enabled us to isolate a live antigen-specific CD4+ T-cells with a simple single cell surface staining after in vitro stimulation for the fast generation of highly specific CD4+ T-cell lines.Our approach offers the striking option to assess the entire pool of CD4+ T-cells with a defined specificity allowing for combined quantitative and qualitative analysis of Th-cell immunity and for isolation of specific Th-cells for targeted cellular immunotherapies. Excessive Innate Immune Responses Could Prime Massive Hepatocyte Apoptosis Induced by Lipopolysaccharide. 1 1 The Third Department of Internal Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan.Background/Aims: Although liver diseases are known to be exacerbated by bacterial infections, the mechanism is still unclear. In a mice model, P. acnes -primed mice show granuloma formation in response to the bacteria, and massive hemorrhagic liver injury is observed after lipopolysaccharide (LPS) injection. However, whether antigen-specific response or granuloma formation is requisite for the injury is unknown. We, therefore, examined whether antigen-nonspecific accumulation of DCs and macrophages in the liver by the overexpression of GM-CSF could prime severe liver injury after LPS injection.Methods: We injected a recombinant adenovirus encoding GM-CSF (AdGM) containing 1Â10 8 plaque-forming units, and one mg per mouse of LPS was administered seven days later into C57BL/6 (B6) mice via a tail vein. The composition of hepatic mononuclear cells were analyzed by flow cytometry. The concentrations of GM-CSF, TNF-a and IFN-g in the serum of mice were measured by ELISA. Liver histology, serum alanine aminotransferase (ALT) levels and apoptosis of hepatocytes were also examined. To further examine the role of Fas pathway and TNF-a in the apoptosis of hepatocytes, we used FasL-deficient mice B6-gld/gld. In both B6-wild type and B6gld/gld mice 100Ag per mouse of a neutralizing anti-TNF-a antibody was intravenously injected 30 minutes before LPS injection.Results: Liver histology of the AdGM-primed mice showed marked infiltrates of mononuclear cells (CD11b + macrophages and CD11c + I-A b+ CD11b + myeloid DCs) without granuloma formation on day 7. Expression of toll-like receptor-4 on intrahepatic mononuclear cells isolated from AdGM-primed mice was up-regulated. Although serum ALT levels were within normal range before LPS injection, the levels were elevated as early as 6 hours after LPS injection only in AdGM-primed mice. The peak levels were 5922 F 3678 IU/L reached at 12 hours after LPS injection, and all those mice died within 24 hours. Serum TNF-a concentrations were elevated after LPS injection, and peaked at 2-6 hours after LPS injection. Hemorrhagic liver injury was histologically recognized, In AdGM-primed mice, TUNEL-positive hepatocyte nuclei were already observed one hour after LPS injection when liver injury was not histologically apparent, and the rates of TUNELpositive apoptotic hepatocytes were increased to about 80% three hours after LPS injection. When AdGM and LPS were injected in FasL-deficient B6-gld/gld mice, serum ALT levels were not elevated by the pretreatment with a neutralizing anti-TNF-a antibody.Conclusions; Our present study provides a new model of severe liver injury, in which massive accumulation of innate immune cells such as DCs and macrophages in the liver by overexpressing GM-CSF enhances the susceptibility to LPS, leading to hemorrhagic liver injury with massive hepatocyte apoptosis after LPS injection. Both TNF-a and Fas-mediated signaling were thought to be important for the hepatocyte apoptosis. Introduction: Neonatal sepsis is a life-threatening disease with an incidence of 1 to 10 per 1000 live births and a mortality rate of 15% to 50%. The clinical signs are nonspecific and indistinguishable from those caused by a variety of neonatal no infective disorders. The aim of this study was to determine sensitivity, specificity, positcive and negative predictive value of neutrophil markers, in early diagnosis of neonatal sepsis.Methods: In this study were comprised 65 neonates with a gestational age of 27 to 38 weeks who suspected for sepsis within 28 days of life. 1 ml of whole blood was obtained from neonates to determine CD64 expression of peripheral blood neutrophils by flow cytometry. Neonates were classified into two groups. In the sepsis group (n = 8) all of neonates were positive blood culture and had clinical symptoms. 12 healthy term neonates with physiologic hyperbilirubinemia classifed in control group.Results: CD64 was elevated in sepsis group and this increase was significant(pb0.001).specificity and sensitivity of CD64 were 100% and 92% respectively. The negative and positive predictive value of CD64 for identifying sepis were 100% and 88% respectively.Discussion: High sensitivity of CD64 (100%) and specificity of 92.3% indicates that evaluation of CD64 as neutrophil marker can help clinicians for early diagnosis of neonatal sepsis. Objective: WhippleTs Disease (WD) is a rare systemic infectious disease prefaced by replication of the actinomycete Tropheryma (T.) whipplei in macrophages of the intestinal mucosa. The possibility of a predisposing immune deficiency is discussed for WD and a reduced TH1 reactivity that could explain the rarity of the disease despite the ubiquitous existence of the pathogen, was already shown for WD patients. However, due to a lack of bacterial preparations T. whipplei-specific intestinal or peripheral immune reactions of WD patients were not analysed till now. T. Whipplei Specific Immune Reactions Methods: T. whipplei-specific stimulations were performed in peripheral blood and lamina propria lymphocytes (LPL) with sonicated T. whipplei cultures, followed by FACS-analysis of expression of cytokines (IFNg, IL-4, IL12) and activation markers (CD69, CD154) of CD4+ T-cells. Several common bacterial and viral antigens (Tetanus Toxoid, Cytomegalovirus and Tuberculin) were used to control general antigen-specific reactivity.Results: We established specific stimulations with recently evolved laboratory strains of T. whipplei and compared for the first time immune reactions of WD patients, healthy controls (agematched and young), and patients with Tuberculosis as a mycobacterial model infection. In every control and patient with Tuberculosis analysed so far, we were able to detect T. whipplei-specific CD4+ T-cells in the blood as well as in the duodenal mucosa. However, WD patients showed a significantly reduced reactivity against the pathogen. The reactivity against SEB of WD patients was slightly reduced compared to controls, whereas the reactivity to common antigens was comparable. Additionally, we used a system with in vitro generated autologeous dendritic cells loaded with T. whipplei antigens that were able to enhance reactivity in healthy controls but still could not induce antigen-specific reactions in WD patients.Conclusion: Hence we can conclude that T. whipplei-specific reactivity occurs in healthy controls and that frequent exposure to the bacteria seems to activate immune reactions, whereas our results hint at a significantly reduced antigen-specific reaction of WD patients resulting in insufficient protection against the pathogen and onset of WD. Adoptive Transfer of CFSE-Labeled Autologous PBMC for the Visualization of T Cell Migration in a Non-Human Primate Model. D. Kunkel, 1 V. Moos, 1 C. Stahl-Hennig, 2 F. J. Kaup, 2 M. Zeitz, 1 T. Schneider. 1 1 Medical Clinic I, Charite-Campus Benjamin Franklin, Berlin, Germany; 2 German Primate Center, Gottingen, Germany.Background: Several inflammatory pathological conditions at mucosal surfaces are thought to be the consequence of altered T cell homing. Our aim was to to study the migration of Tcells under such conditions in the non-human primate system. The transfer of allo-or congenic fluorescently labeled cells has provided a useful technique for the visualization of T cell activation, proliferation and migration in vivo and is a well established application in the mouse model. However, until now there is no equivalent technique in the non-human primate system, which for several diseases is the only available animal model where the pathologic development is similar to that in humans.Methods: Here we describe the establishment of an autologous transfer system in the rhesus macaque model. Mononuclear cells were isolated from peripheral blood, labeled with carboxyfluorescein diacetat, succinimidyl ester (CFSE) and tracked after reinjection.Results: Flowcytometric analysis after transfer revealed a distinct population of labeled lymphocytes in peripheral blood and lymph nodes as well as in the intestinal mucosa of all animals. The percentage of labeled T cells in peripheral blood remained almost stable for up to 4 weeks, whereas the fluorescence intensity of the whole population decreased slightly, probably due to physiological turnover of intracellular proteins. Preliminary results indicate an increased frequency of CD4+ T cells migrating to the mucosa after transfer of in vitro activated cells compared to the migration of non-activated cells.Conclusions: The well established transfer of CFSE labeled cells in the mouse system has been successfully adapted to the rhesus macaque system and allows us to analyse the migratory behaviour of T cells in diseases where the only available animal model is the non-human primate system, e.g. Psychological stress is known to affect immune function and to influence on infectious disease susceptibility. Previous studies have demonstrated that chronic stress can impair humoral immune response to influenza vaccination but no data is available on posttraumatic stress disorder (PTSD). PTSD, according to Diagnostic and Statistical Manual of Mental Disorders (DSM-IV), is a condition (or anxiety disorder) that can occur after exposure to extreme traumatic experience and is accompanied by intense fear, helplessness or horror. Exposure to trauma can result in immune deregulation, and increasing number of evidence suggests that there are immune alterations associated with PTSD.The aim of this study was to determine the effect of psychological stress on the immune response to influenza vaccination in war related PTSD patients (n = 32). Peripheral blood mononuclear cells (PBMC) and sera were obtained before and 14 days after vaccination (Agrippal, Chiron, Italy) from patients and control subjects during 2003/2004 winter season. Detection of specific antiviral antibody titre in sera for all viral strains contained in the vaccine was performed with inhibition of hemagglutination (IH) assay. Ex vivo tetramer staining of recently activated CD8+ T lymphocytes was used for monitoring of T cell response specific for HLA-A*0201restricted influenza A matrix antigen (M1 58-66 ) and haemaglutinin antigens (A/New Caledonia/H1N1, HA 345-354 , HA 542-550 ) before and after influenza vaccination. Sixteen patients showed 4-fold increase of H1N1 antibody titre 14 days after vaccination. In four of total ten HLA-A*0201+ patients 2-to 4-fold increase of frequency of recently activated influenza-specific T cells was observed after vaccination. However, PTSP patients showed diminished frequencies of influenza-specific CD8+ T cells after vaccination compared to healthy controls. Generated humoral response in our patients argues against the hypothesis that post-traumatic stress might influence the protection following vaccination. Diminished cellular response in PTSD patients could indicate that immune dysregulation observed earlier in these patients selectively affects cellular immune response. Cholesterol Loading of T Cells as a Model of T Cell Aging. A. Larbi, 1,2 G. Dupuis, 2 C. Fortin, 1 Lymphocytes functions are impaired in human aging explaining the increased susceptibility to diseases. It was observed that defects in T-cell receptor signal transduction could explain this age-related immune senescence. Lipid rafts are critical components of the membranes that serve for signal transduction enhancement. Our hypothesis is that changes in lipid rafts properties could explain defects in signal transduction. Cholesterol is a major component of the cell membrane and especially of lipid rafts. In this connection, we found an elevation by two folds of lipid rafts associated-cholesterol in T-cells with aging. We sought to determine the role of cholesterol in immune senescence. We modulated cholesterol content in peripheral T-cells from normolipemic young donors using a mixture of cyclodextrin and cholesterol. Cholesterol content in whole cell extracts as well as in lipid rafts was measured by HPLC. We were able to increase cellular cholesterol content by 1.5 fold to up to 7 folds. We observed changes in cell shape and size (microscopy, flow cytometry) as well as a dramatic loss of membrane fluidity. We analyzed T-cell proliferation and protein phosphorylation (western-blotting) following CD3 and CD28 stimulation. We found that an in vitro increase in membrane cholesterol by 2-folds have similar consequence on signal transduction as observed in human aging. We could here establish a T-cell model of aging by cholesterol modulation. Altogether, these data suggested that signal transduction critically depends on membrane cholesterol content and that changes in lipid rafts properties may be taken into account to explain immune senescence as well as in other pathological diseases. Programmed death-1 (PD-1) and its ligands PD-L1 and PD-L2 are members of B7/CD28 superfamily, and play important roles in modulating the adaptive immunity in experimental autoimmune encephalomyelitis (EAE), type-1 diabetes, asthma and transplantation. In the study of PD-1 pathway in EAE, we found that multiple injections of either PD-1 or its ligand blocking antibody after standard EAE induction caused a shock syndrome in multiple mouse strains, which included 129X1/SvJ, C57BL/6, and SJL/j mice. Animals showed piloerection, cyanosis, drop in body temperature and were eventually dead within 2 hours of antibody injection. Interestingly, BALB/c mice, a strain known to be TH2 prone, are relatively resistant to the development of shock syndrome. In addition, the blockade of PD-1 pathway did not enhance OVA peptide induced anaphylaxis in BALB/c mice. Pathological study in shock mice found massive neutrophil infiltration in the lung, liver and skin, suggesting PD-1 pathway blockade directly or indirectly activated neutrophils in this model. Consistently, freshly isolated neutrophils from normal mice were found to express PD-1 and PD-L1, and neutrophils isolated from anti-PD-L1 treated mice showed enhanced and accelerated superoxide production upon PMA stimulation. We are currently further studying the role of PD-1 pathway in animal models known to involve neutrophils in pathogenesis, such as sepsis and ischemiareperfusion injury. T helper lymphocytes play a major role on infectious diseases and these cells can be driven into at least two different subpopulations according to their cytokine production pattern. Th1 lymphocytes are implicated on the resolution of infections caused by intracellular pathogens such as Leishmania species, while Th2 cells are thought to be important to the establishment of these infections. Based on this knowledge ELISA tests were performed to measure the plasma levels of anti-Leishmania specific IgG1, IgG3, IgG4 and IgE antibody isotypes. Production of Interleukine-4 and IFN-g by peripheral blood mononuclear cells (PBMC) from subjects living in an endemic area of American Cutaneous Leishmaniasis (Bahia State, Brazil) was analysed. Patients were grouped on active lesions (n = 20), cured lesions (scars) (n = 40) and asymptomatic (n = 34). PBMC were cultured for 24 and 96 hours stimulated with Leishmania braziliensis antigens. The analysis of IgG isotypes response showed that both IgG1 and IgG3 were able to differentiate the group of patients presenting active lesions from the others (Kruskal Wallis; P = 0.0004 and P = 0.0004, respectively). Moreover, the analysis of cytokine production revealed that IFN-g level was significantly higher on patients with active or cured leishmaniasis than on the asymptomatic group (Kruskal Wallis; pb0,0001). There was a correlation between the production of IFN-g by PBMC stimulated with L. braziliensis antigens and the plasma levels of IgG1 and IgG3 antibody isotypes in patients presenting active lesions (Spearman Correlation; P = 0.0065 and P = 0,0107, respectively). The level of IL-4 was significantly higher on patients presenting active lesions than on the others (Kruskal Wallis; P b 0.0001). The results confirm that IFN-g is produced during human Cutaneous Leishmaniasis and that, during active lesions, a concomitant high production of IL-4 is observed. Our results also point out that IgG1 and IgG3 are useful antibodies to differentiate patients with active lesions. Investigatin of IL-4 Gene Polymorphism in Patients with Brucellosis. 1 Brucellosis is endemic in many Middle East countries including Iran, where it undermines animals and human health. The Immune response against Brucella involves both humoral (Th2) and cell-mediated (Th1) immunity. The Th1/Th2 cytokine ratio seems to be involved in the susceptibility or resistance to Brucella infection. Th1 cytokines confer resistance were as Th2 cytokines predispose to brucellosis. IL-4 is a Th2 cytokine which its production can be affected by its gene polymorphism at position -590(C/T). The aim of this study was to investigate the association between IL-4 gene promoter polymorphism and susceptibility to brucellosis.One hundred and ninety seven patients with brucellosis, 186 healthy individuals who were members of patients family and 82 healthy animal husbandmen who had animals involved with brucellosis, were included in this study. All individuals were genotyped for IL-4 gene promoter polymorphism at position -590(C/T) using PCR-RFLP.The results showed the distribution of CC genotype is significantly more in animal husbandmen than the patient group (P = 0.034). No statistical significant differences in genotypes distribution were shown between patients and their healthy family, but there was a trend toward increased frequency of CC genotype in family group (P = 0.063).As data shown, the distribution of CT and TT genotype which were associated with more production of IL-4 were increased in patients (29.3%) compare to animal husbandmen (17.4%, P = 0.034)) and patientsT family members (21%, P = 0.063). We suggest that individuals who carry T allele can produce more IL-4 and this cytokine induce Th2-type immune response which could be important to develop a full-pictured brucellosis. While, individuals with CC genotype can probably induce a Th1-type immune response more effectively and control the Brucella before developing the disease. Immunophenotype Characterization of Peripheral Blood T Lymphocytes before and after Treatment in Tuberculosis Patients. Fereshteh Sahebfosul, Farzad Oreizi, Zohre Pessaran, Ahmad Ghavaminejad. 1 Immunology, Medical School, Isfahan, Isfahan/ Isfahn, Islamic Republic of Iran; 2 Immunology, Medical School, Isfahan, Isfahan/Isfahan, Islamic Republic of Iran; 3 Immunology, Medical School, Isfahan, Isfahan/Isfahan, Islamic Republic of Iran; 4 Immunology, Medical School, Isfahan, Isfahan/Isfahan, Islamic Republic of Iran.Intoduction: Tuberculosis is a chronic mycobacterial infection. The main effecter cells against mycobacterium tuberculosis are CD4 + T lymphocytes.Objective: Our objective in this research was to evaluate the quantity of T lymphocytes and their subpopulation before and after treatment with combination of 4 drugs recommended by WHO (DOTS Protocol) in sputum-positive tuberculosis patients.Materials and Methods: 20 patients as cases and 20 healthy people were selected as controls. Flow cytometry was done for TCD3+, TCD4+ and TCD8+ lymhocytes by use of monoclonal antibodies.Result: Our results showed that there was a defect in cell mediated immunity during tuberculosis showing itself as decrease in TCD3+ and TCD4+ lymphocytes and increase in TCD8+ lymphocytes. The changes in TCD3+ and TCD4+ but not in TCD8+ were reversible after 2 months of treatment.Conclusion: The result of our study and other studies confirm defect in cell mediated immunity during infection with mycobacterium tuberculosis. Although decrease in CD4 + lymphocytes that are main cells in defense against tuberculosis, has been established, there is no consensus about decreasing or increasing of CD8+ lymphocytes in tuberculosis. Immunity to T. cruzi is complex, minimally involving a substantial antibody response and the activation of CD4+ and CD8+ T cell responses. Class I Restricted Epitopes Highly We have recently shown that chronic chagasic patients with mild clinical disease have a significantly higher frequency of IFN-g producing T cells specific for a parasite lysate than do individuals with severe disease. However, it has been difficult to identify the specific epitopes recognized by T. cruzi-specific T cells, and thus to quantify the response of CD8+ T cells from infected individuals. In T. cruzi-infected mice, peptides from transsialidase family proteins (ts), are dominant targets of the CD8+ T cell response. In the present study, we have investigated the response of CD8+ T cells from HLA-A2.1+, T. cruzi-infected individuals to ts family, HLA-A2.1-binding epitopes.The 1294 genes annotated as encoding ts family proteins were screened for homologues of two ts-derived, HLA-A2.1-binding peptides that we had previously found to be recognized by a low frequency of CD8+ T cells in HLA-A2.1+ infected subjects. This screening yielded a total of 845 homologues, 71 of which were encoded by N50 ts. Thirty-two of the 71 peptides elicited IFN-g production by CD8+ T cells from HLA-A2.1 transgenic mice chronically infected with T. cruzi, with peptides with the highest frequency of occurrence and the highest predicted HLA-A2.1binding affinity stimulating the greatest response. Antigenicity of ts peptides was fairly associated with the capacity to bind multiple molecules from the A2 supertype. To ascertain whether these epitopes were recognized by CD8+ T lymphocytes from T. cruziinfected subjects. IFN-g ELISPOT responses were evaluated with PBMC from chronic chagasic patients.IFN-g secretion was demonstrated in 10 out of 17 patients (59%) with mild disease, with 24 out of the 28 peptides assayed recognized by at least one patient. For each responder, the frequency of peptides that were capable of eliciting recall IFN-g responses varied between 11% (3 of 28 tested) and 100% (5 of 5 tested); peptides ts3, ts37, ts38, ts44 and ts66 being the most frequently recognized. By contrast only 2 out of 10 patients with severe clinical stages showed positive responses. These data demonstrate that the frequency of IFN-g producing T cells in chronic chagasic patients is determined in part by the clinical status of the patient and also by the frequency of occurrence of the epitopes in the T. cruzi genome. We have identified a very specific set of class I MHC restricted peptides target of immune responses in patients capable of controlling T. cruzi infection. The identification of CD8+ T cell targets in the natural infection with T. cruzi may be applicable to the development of vaccines against T. cruzi. Human Parvovirus B19 (dB19T) is a ubiquitous DNA virus that causes erythema infectiosum, polyarthropathy, transient aplastic crisis and fetal death. IFN-gamma elispots, intracellular cytokine staining, and HLA-peptide tetrameric complex (dtetramerT) staining were used to identify CD8+ T lymphocyte responses to the non-structural protein NS1. To understand the origins of such responses and their potential role in disease we prospectively analysed the evolution of virus-specific CD8+ T cell responses, in five individuals, during and after acute infection. Individuals responded to B19 tetramers at frequencies up to 2% CD8+ T cells. Surprisingly their responses increased over the first year post infection despite resolution of clinical symptoms and control of viremia. Parvovirus-specific CD8+ T cells rapidly developed and maintained an activated CD38+ phenotype, with strong expression of perforin and CD57 and downregulation of CD28 and CD27. These cells possessed strong effector function and intact proliferative capacity (demonstrated by IFN-gamma elispots, Chromium-51 release assays and tetramer staining of B19-specific CD8+ T cell lines). In remotely infected individuals lower frequencies of cells specific for individual epitopes were observed, typically b0.5% of CD8+ T cells, which were CD38 low although also CCR7 low. The likely explanation for the striking persistent expansion of such activated mature effector cells after acute resolution of infection-analogous to CMV infection-is through low level antigen exposure. Such cells may contribute to the long term control of this significant pathogen. There is substantial evidence that BCG provides partial protection against pulmonary tuberculosis. But this evidence does not convincingly demonstrate that BCG prevents tuberculosis (TB) development. The availability of Mycobacterium tuberculosis H37Rv genome sequence has thrown open new opportunities for designing a rational vaccine for TB. The identification of T cell epitopes from immune relevant antigens remains a critical step in the development of vaccines. The discovery of two multigene families, PE and PPE, which make up to 8% of Mycobacterium tuberculosis H37Rv genome has kindled an interest in using these proteins as potential vaccine candidates. All possible nonameric peptide sequences from PE and PPE proteins were analysed computationally, for their ability to bind to 33 alleles of class I HLA. Of all PE and PPE proteins, a significant number of these peptides are predicted to be high affinity HLA binders, irrespective of the length of the protein. Two proteins from PE and PPE family of Mycobacterium tuberculosis genome have been selected for further study as these proteins show high percentage of binding peptides. The recombinant proteins were tested for their ability to elicit immune response in mouse model. The recombinant Rv1818c is good inducer of antibody response but very poor inducer of T cell response, where as Rv3812 is shown to induce good T cell response in terms of invitro T cell proliferation, IFNg secretion and DTH response. The predicted epitopes are experimentally verified by identification of T cells, which specifically recognize the naturally processed epitope in an HLA-restricted fashion. The Study between the Changes of CD35 Expressed on Erythrocyte in Patients with Hepatocirrhosis and the Severity of Liver Function Destroying.Ma Chi, 1 Wang Haibin. 2 [Objective] To study the relationship between the changes of CD35 expressed on erythrocyte (ECD35) in patients with hepatocirrhosis and the severity of liver function destroying. [Methods] One-step detection method of ECD35 has been used 108 cases in hepatocirrhosis as followed, 2 percent of Glutaraldehyde-fixed red blood cells (RBC) are analyzed in V well microstate plates. To the cell buttons in each well, the compound of b-CR1Ab+SA*AP was added and mixed thoroughly. The supernatant was transferred to a clean U microtitre plate to facilitate reading in a plate reader at 405nm (A 405 ). The number of CD35 was calculated by the standard of red cell. Another detection of ALT, CHE, ALB, GLO, TBIL, and GAM were assayed with the machine of AU 5400. [Results] The number of ECD35 in patients with Hepatocirrhosis was significantly lower than that in healthy individuals. The patients were divided in to two groups according to the levels of ECD35 by 200/cell. The results showed that the patients with low activity of CHE and decreased ALB were almost in low CD35 number groups, the levels of GLO, TBIL and GAM are increased accordingly. There is no obviously relationship between the changes of ALT and ECD35. [Conclusions] The results suggest that the quantity of CD35 on erythrocytes may be involved in pathogeny, development and prognosis of the hepatocirrhosis. The detection of the Quantity of ECD35 maybe used as an important assay in liver functions Introduction: We have identified a group of patients with recurrent respiratory infections who have normal total immunoglobulins and normal response to protein antigens but who failed to develop IgG antibodies to the Pneumococcal 7-valent conjugate vaccine (PCV7) serotypes. Methods: This is a retrospective study of 60 patients referred to our clinic for evaluation of recurrent respiratory infections who received complete immunization with the PCV7 vaccine for age, according to ACIP guidelines. All patients were evaluated with total immunoglobulin and IgG subclass concentrations and their immunization history was documented. We measured IgG and IgM anti-pneumococcal polysaccharide (PP) antibody titers against serotypes 1, 4, 6B, 9V, 14, 18C, 19F and 23F by a standardized ELISA test. The data were analyzed using Epi Info and SPSS. Samples were obtained from patients who were already immunized by their primary care physicians. The patients were assembled into 3 groups, an unimmunized group with laboratory data prior to the vaccine and two immunized groups of responders and nonresponders according to their IgG anti-PP antibody titers. Nonresponder patients were defined as those with IgG anti-PP antibody titers to all 7 serotypes included in the PCV7 vaccine not statistically different from unimmunized controls. Results: Despite differences in IgG anti-PP antibody titers, both responder and nonresponder patients had IgM antibody titers to all 7 serotypes significantly higher than those of unimmunized controls. The cellular and molecular mechanisms underlying this defect are under investigation. A Delayed Diagnosis of Cystic Fibrosis in a Patient with Asthma and Allergic Bronchopulmonary Aspergillosis. 1 1 Allergy/Immunology, Walter Reed Army Medical Center, Washington, DC, USA. INTRODUCTION: Allergic Bronchopulmonary Aspergillosis (ABPA) is a complex hypersensitivity reaction including both IgE-and IgG-mediated immune responses following Aspergillus colonization of an asthmatic airway. Cystic Fibrosis (CF) is a multisystem disease characterized by disordered exocrine gland function. ABPA is extremely difficult to recognize in the context of CF, as clinical, radiographic, and immunologic features frequently overlap.The role of oxitative stress in the pathogenesis of pancreatitis and benefits of antioxidants have been suggested in various studies. Superoxide dismutase (SOD), a primary antioxidant enzyme, scavenges reactive free radicals by catalyzing the dismutation of superoxide anion into molecular oxygen and peroxide. Copper and zinc-containing SOD (Cu/Zn SOD) is the intracellular eznymes, which activity depends on metals.It has been shown that metallotionein (MT), also antioxidant protein, in high concentration in the pancreas-serves a function of exceptionally sensitive indicator of Zn status. Our recent studies confirm that MT is present in exocrine and endoxrine cells of patients with chronic pancreatitis, particularly in acinar cells of pancreas (1) .In the last years, evidence was provided that there appears to be a decrease in Cu/Zn SOD expression in pancreatic cells from normal pancreas to chronic pancreatitis (2) .AIMS & METHODS:The aim of the study was to identify immunohistochemically the distribution pattern of the Cu/Zn SOD in chronic pancreatitis (CP) and chronic exacerbated pancreatitis (CEP). Samples of tissues of normal pancreas (n = 5) (obtained at autopsy) and CP (n = 14), CEP (n = 2) were verified histopathologically and then Cu/Zn SOD was localized by immunohistochemical staining using the primary polyclonal anti-human Cu/Zn SOD antibody (Calbiochem, UK) and second anti-sheep peroxidase conjugated antibody (Sigma, Germany) to visualize Cu/Zn SOD-Ab complexes. CASE REPORT: A 49 year old Caucasian female with a past medical history of steroid-dependent asthma, allergic rhinitis, ABPA, and frequent pneumonias presented with acute maxillary sinusitis. Past medical history was additionally remarkable for infertility and intermittent episodes of hemoptysis. At 26 years of age Mycobacterium intracellulare pneumonia was diagnosed with symptoms of respiratory distress and hemoptysis. Two years later, APBA was diagnosed. In the subsequent 23 years, the patient had multiple hospitalizations for basthmaQ exacerbations, hemoptysis, and pneumonia. Daily oral steroids and intermittent courses of antibiotics were given to control asthma symptoms and infections. At 43 years of age, the patient was hospitalized for Pseudomonas pneumonia with symptoms of respiratory distress and hemoptysis. At 49 years of age, while presenting with acute sinusitis, a chest and sinus CT were performed. Sinus CT was remarkable for chronic sinusitis changes, and chest CT demonstrated multifocal bilateral cylindrical bronchiectasis, with multiple mucous-inspissated dilated bronchi. Sweat chloride testing and genetic testing were consistent with the diagnosis of CF with the DF508 and L206W mutations. It further demonstrates the importance of clinical suspicion for CF in patients of all ages who present with a history of asthma and frequent sinopulmonary infections. Sweat chloride testing is a non-invasive testing modality that may dramatically alter both management and prognosis for patients with CF. Determination of Borrelia burgdorferi Outer-Surface Protein A 161-175 Peptide Binding to HLA-DR Molecules Associated with Antibiotic-Refractory or Antibiotic-Responsive Lyme Arthritis. Objective: Clinical correlations have linked antibiotic-refractory Lyme arthritis with certain HLA-DR molecules and the B. burgdorferi T cell epitope, outer surface protein A 161-175 (OspA 161-175 ). Previously, HLA-DRB1*0401, 0101, and 0404 molecules, which are associated with antibiotic-refractory arthritis were shown to bind this peptide, whereas the DRB1*0801 and 1101 molecules, which are associated with antibiotic-responsive patients, did not. To further these studies, we determined the relative binding affinity of OspA 161-175 to 14 HLA-DR molecules, and correlated peptide binding with MHC frequency in antibioticrefractory or antibiotic-responsive arthritis patients.Methods: The cDNAs of the extracellular domains of DRa and DRh attached to the leucine zipper sequences were cotransfected and purified in Drosophila cells. DR molecules were purified by affinity chromatography. Dilutions of the peptide (0.0001-50 AM) were tested, and the half maximal binding concentration (1/2 max) was determined. The DRB1*0401 molecule bound the peptide strongly (1/2 max = 0.003 AM), the DRB1*0101, 0404, or 0405 molecule bound it moderately well , and the DRB1*0402 or 0102 molecule bound it weakly (1/2 max = 4-6 AM). In addition, testing of the linked and equally expressed DRB1*1501/DRB5*0101 molecules showed that this DRB5 molecule, but not the DRB1 molecule, bound the peptide moderately well. Altogether, 79% of patients with antibioticrefractory arthritis had at least one of these 7 OspA peptide-binding HLA-DR molecules compared with 46% with antibiotic-responsive arthritis (odds ratio = 4.4, Pb0.001). Moreover, patients with two OspA 161-175 -binding alleles were 9.6 times more likely (CI 1.9-46.9) to have an antibiotic-refractory course than patients with two non-OspA 161-175 binding alleles.Conclusion: The highly significant correlations of OspA 161-175 peptide binding to implicated HLA-DR molecules further supports the hypothesis that T cell recognition of this bacterial epitope is important in the autoimmune pathogenesis of antibiotic-refractory Lyme arthritis. This is the only form of chronic inflammatory arthritis in which the identity of each component of the diseaseassociated tri-molecular, MHC-peptide-TCR complex has been deciphered. Cell migration and adhesion are both important for controlling mycobacterial infection and are critically dependent on the reorganization of the cytoskeleton. Mycobacteria elicits rapid morphological changes such as cell spreading the process relevant to in vivo changes of macrophage shape during extravasation and migration. In this study we investigated the BCG mycobacteriainduced signaling events leading to macrophage cytoskeletal rearrangements employing specific pharmacological inhibitors to suppress distinct kinase pathways known to be elicited by infection. Viable or lysed mycobacteria, as well as purified cell wall lipoprotein p19, TLR2 agonist, induced RAW264.7 cells to extend actin-rich pseudopods which imparts circular spreading within 30 min that was substituted by persistent cell polarization 24h post-treatment. BCG induced rapid phosphatidylinositol 3-kinase, PI3-K, activation which become recruited to the activated TLR2 receptor. Suppression of PI3-K with LY294002 inhibitor abrogated generation of cell protrusions and polarity demonstrating the involvement of PI3-kinase pathway in actin reorganization essential for cell motility. Inhibition of MEK1/ERK kinases with PD98059 reduced the number of polarized cells but did not prevent filopodia or lamillopodia formation suggesting the role of this pathway in stabilization of leading lamillopodia. Nor NFn B nor p38MAPK activation by BCG were important for cytoskeletal rearrangements observed although their suppression inhibited chemokine and growth factors secretion by activated macrophages that could promote the cell motility in an autocrine manner. h 1-integrins blockade with a corresponding antibody inhibited macrophage spreading and polarization but had no effect on pseudopod protrusions or PI3-K activation demonstrating the down-stream position of integrin-mediated adhesion in signaling pathway leading to motility phenotype.The data obtained demonstrate that direct effect of mycobacteria on macrophage shape at least in part is mediated through TLR2-dependent PI3-K activation.Financial support: CNPq, FAPERJ, CAPES. Chlamydia pneumoniae (Cpn) is an obligate intracellular bacterium known to cause acute upper respiratory tract infections, but recent studies show Cpn is also involved in a variety of chronic inflammatory diseases, including atherosclerosis and possibly autoimmune diseases like multiple sclerosis as well as arthritis and asthma. In fact, we have reported that this organism can be detected by PCR for Cpn specific 16s r RNA in human peripheral blood mononuclear cells associated with subclinical chronic bacterial infection. Even though this organism grows preferentially in epithelium cells in the respiratory tract, in the present study we show that these bacteria can infect and replicate in the human monocytic cell line (THP-1) and the human T cell line (Molt-4) and also human peripheral blood mononuclear cells in vitro as detected by FITC-conjugated anti-Chlamydia monoclonal antibody (specific to Chlamydia LPS) staining or PCR for Cpn 16s rRNA. The Cpn mRNA level was upregulated at 48h as compared to 24h after infection assessed by real-time PCR for Cpn 16s rRNA ( p b 0.05). We also found that Cpn infected cells evinced marked alteration of cytokine production important in immune responses to microbial infection. Cpn infection of human immune cells modulated a variety of cytokines. In particular, Cpn infected MOLT-4 and THP-1 cells suppressed TGF h1 production as compared to uninfected cell assessed by ELISA ( p b 0.05). In contrast, Cpn infection of the human cell lines in vitro markedly stimulated production of proinflammatory cytokines, including TNF a and IL 10. Interestingly Cpn infected PBMCs enhanced production of TNF a, IFN g, IL 4 (PHA induced), IL 10 and IL 12 as compared to lower TGF h1. Infection with viable Cpn stimulated more IL 12 and IFN g(Th1 cytokines) and less IL 4 (Th2 cytokines) as well as TGF h1 (Th3 cytokines) compared to stimulation with heat killed Cpn in PBMCs. This may be related to antibacterial immunity by this common opportunistic bacterium. Further studies are warranted to assess the role of Chlamydia on immune responsiveness, since Cpn are considered important in induction and progression of atherosclerosis, neurologic or autoimmune diseases. The cytokine profile differences observed appeared related to antibacterial immunity and perhaps autoimmunity. Thus, infection of human immune cells with Cpn may result in significant immune deregulation.Sa2.56. Homeostatic Chemokines: Organizers of Cellular Interactions Required for T Cell Priming in the Pulmonary Environment during the Influenza Infection.in secondary lymphoid organs (SLOs). Additionally, at early embrionary steps of the development of these tissues, they organize the recruitment of inducer cells (CD3-CD4+CD45+), facilitating their interaction with organizer populations (VCAM+ICAM+). This cellular event promotes a second wave of chemokine production, responsible for the latter recruitment of immune cells to their future homes in B-and T cell zones. Additionally, HCs have been also detected at sites of inflammation, where they contribute to the formation of ectopic tertiary lymphoid structures. Rationale: in our murine model of influenza infection, we detected HC local production by Northern blot, Taqman-PCR and immunohistochemistry. This finding encourages us to explore the participation of these molecules during the pulmonary immune response against influenza viruses. Methods: C57BL/6-, cxcl13 -/-, plt/plt -/-and lt alpha -/-mice were infected with PR8 influenza virus and sacrificed at different time points, collecting spleens and lungs for the analysis of the immune response and the course of the infection. Cellular interactions and distribution of immune cells was evaluated by immunohistochemistry and immunofluorescence. Finally, by using flow cytometry, we determined the phenotype of immune cells, the functionality of CD8 T cells and the migration of T and B-lymphocytes to the spleen of uninfected mice. Results: we found that the absence of homeostatic chemokines is highly associated to the generation of altered cellular interactions in the local environment, which modified the course of the infection. It appears that their production is playing an important role in the organization of the B-and T cell microenvironments in the lung, in a similar way than they do in SLOs. Interestingly, it was appreciated that in the absence of the draining lymph node (cxcl13 -/-) or adequate APC-T cell interactions (plt/plt -/-), influenza-infected mice were still able to mount an adequate primary T cell response, killing the vast majority of viruses. Conclusions: Our findings reinforce the idea that the lung can work as an alternative place for T cell priming and that local cellular interactions could be enough for the initiation of the T cell response against Influenza viruses. T cells are heterogeneous, varying in terms of their phenotype, functional capabilities and history of antigen-encounter. The derivation of a functionally relevant model for classifying CD4+ T cells has been hampered by limitations on the numbers of parameters that may be measured using classical 4-colour flow cytometry. The purpose of this study was to develop a detailed, functionally meaningful scheme for classifying human CD4+ T cells. To achieve this we have taken advantage of the introduction of reagents for 5-colour flow cytometry and have performed multi-colour FACS analysis on resting, polyclonally-activated and antigen-stimulated preparations of peripheral blood mononuclear cells taken from healthy individuals. We show that CD4+ T cells are predominantly distributed between six of eight possible subsets identified by expression of CCR7, CD45RA and CD28.One subset corresponds to the previously described naive compartment and one has the phenotypic properties of the described central memory compartment. Two of the remaining four subsets include antigen-experienced CD4+ T cells that express CD45RA. We find clear differences between CD4+ T cells within the different subsets in terms of their expression of cytolytic mediators, their capacity to secrete cytokines and their ability to proliferate. Furthermore, we find that T cells specific for different virus infections accumulate within different subsets, implying that protective immunity against different pathogens involves CD4+ T cells with different phenotypic and functional properties. On the basis of these results we propose a crosssectional model for classification of peripheral CD4+ T cells. Knowledge of where T cells lie on this model informs about their functional capacity and can reflect their history of antigenexposure. The objective of this work was to characterize directly the Th1 (IL12p70, IFN-g), Th2 (IL4, IL10) cytokine patterns induced by spleen cells from BALB/c mice presensibilized by intranasal route and restimulated with diphtheria toxin Cry1A mutants. Differential cytokines induced by spleen cells were measured and quantified by ELISA. We found that each set of toxins are priming different effectors T cells, measured by. Wild type Cry1A toxins are priming mostly Th1 cytokine producers whereas DTB quimeric toxins are priming both Th1-Th2 cytokine producers cells, suggesting that eight B fragment diphtheria toxin motif exchanged in domain I from Cry1A toxins are influencing the capacity of these toxins to modulate the Th cellular immune response. In addition the data support the assumption that helical topology from domain I contribute to the cellular properties of the Cry proteins by acting like epitopes for T cells subsets and this in agreement with theorethical predictions,using ANTHEPROT program. By other hand it was observed that Cry1A toxins have the ability to induce regulatory T cells. In this aspect Cry1A toxins represent a good model to study several aspects of immunoregulation at the molecular and cellular level in mice. Intranasal Vaccination with Chlamydial Protease-Like Activity Factor and Interleukin-12 Promotes the Clearance of Pulmonary C. trachomatis Infection.Ashlesh K. Murthy, 1 Yu Cong, 1 Guangming Zhong, 2 Bernard P. Arulanandam. 1 1 Biology, University of Texas at San Antonio, San Antonio, TX, USA; 2 Microbiology and Immunology, University of Texas Health Science Center, San Antonio, TX, USA.Chlamydia trachomatis is a gram-negative bacterium that infects the human conjunctiva, respiratory and genital tract, and is a cause of significant morbidity worldwide. Chlamydia has evolved various immune evasion mechanisms that have posed complex problems in the development of an efficacious anti-chlamydial vaccine. HeLa cells infected with C. trachomatis display a progressive reduction in immunostaining for h2-microglobulin (MHC class I), which parallels the accumulation of intracellular Chlamydial protease-like activity factor (CPAF). Therefore, neutralization of CPAF activity in vivo may be a viable vaccine approach against C. trachomatis infection. To this end, we have shown the efficacy of interleukin-12 (IL-12) as a potent mucosal adjuvant. BALB/c mice were immunized intranasally (i.n.) CPAF + IL-12 immunized animals displayed significantly greater reduction (N95%) in lung bacterial load on day 10 after C. trachomatis challenge as compared to mice immunized with CPAF, IL-12 or PBS alone. CPAF + IL-12 immunization induced significantly higher titers of serum anti-CPAF total Ab, IgG2a and IgG2b antibodies as compared to animals immunized with CPAF, IL-12 or PBS alone. Furthermore, pulmonary anti-CPAF total Ab, IgG2a and IgA were induced to higher levels in animals receiving CPAF + IL-12 as compared to other immunization groups. In addition, CPAF + IL-12 immunized MHC class I deficient mice displayed reduced bacterial clearance after pulmonary C. trachomatis challenge as compared to vaccinated wild type animals. Together, these results demonstrate that i.n. CPAF + IL-12 immunization induces robust systemic and mucosal antibody responses and may enhance the clearance of C. trachomatis from the lungs via a CD8+ T cell mediated mechanism. Identification of ssRNA Sequences That Stimulate Innate Immunity through TLRs. Viral infection of the mammalian host triggers the immune system to innate and adaptive immune responses. Recognition of these conserved pathogen-associated molecular patterns (PAMP) is directed mostly by Toll-like receptors (TLR). Ten TLR family members have been reported in human where TLR9, 8, 7 and 3 respond to nucleic acid derivatives and are intra-endosomal in location, potentially allowing for viral PAMP recognition.Recently, we have discovered that single-stranded RNA (ssRNA) stimulates both human and mouse immune cells to secrete cytokines and chemokines. In response to ssRNA human PBMC release IFN-alpha, IFN-gamma, TNF-alpha, IL-6, IL-8, IL-10, IL-12p40, IP-10 and MCP-1 while Th2 cytokines and chemokines were not detected. Monocytes were the main source for TNF production, pDC the main source for IFN-alpha and NK-, NK/T-cells the main source for IFN-gamma production upon stimulation with viral derived ssRNA sequences. We have identified genomic regions and sequences from ssRNA viruses that are stimulatory and can show that these responses are sequence specific. ssRNA sequences are currently under pre-clinical investigation and may serve as potential vaccine adjuvants and immune modulators. The human pathogen M. tuberculosis is responsible for more deaths than any other infectious disease. The public health problems posed by tuberculosis have grown more serious as a consequence of the global AIDS epidemic and the emergence of multidrug resistant strains of M. tuberculosis. To combat this ongoing worldwide scourge, vaccine development for tuberculosis continues to be a global priority. Most infected individuals develop long-lived protective immunity, which is able to control and contain M. tuberculosis in a manner that is T cell-dependent. Thus, there has been considerable interest in understanding how different T cell subsets contribute to the primary immune response following infection, and whether those T cells can be elicited by vaccination and can mediate protection against M. tuberculosis. There is excellent experimental evidence that CD8+ T cells participate in the immune response to tuberculosis, which has generated interest in vaccine strategies that elicit M. tuberculosis-specific CD8+ T cells. To activate CD8+ T cells, mycobacterial antigens need to enter and be processed by the MHC class I pathway, which can be targeted by using DNA vaccines or on live attenuated vaccines strains. Unfortunately, evaluating the role of CD8+ T cells in host resistance and determining whether vaccines elicit protective CD8+ T cells has been hampered because few mycobacterial antigens have been identified that are recognized by CD8+ T cells. Thus, even basic questions concerning the function of CD8+ T cells during M. tuberculosis infection remain unanswered.We have identified an epitope of the CFP10 protein that is recognized by up to 30% of the CD8+ T cells in the lungs of mice following M. tuberculosis infection. Interestingly, deletion of the region of the mycobacterial genome that encodes CFP10 was the original event that resulted in the attenuation of M. bovis BCG and also attenuates M. tuberculosis. Furthermore, most M. tuberculosis infected people have evidence of immunity to the CFP10 antigen. As predicted, CFP10-specific CD8+ T cells were detected in mice infected with the Erdman and H37Rv M. tuberculosis strains, but not in mice infected with H37Ra or BCG. Importantly, CFP10-specific CD8+ T cells recognized M. tuberculosis infected macrophages. We have previously shown that in vivo cytolytic activity of CD8+ T cells specific for mycobacterial antigens could be detected in the spleen, pulmonary LN and lungs of infected mice. We now in the process of determining whether perforin and Rab27a are required for in vivo cytotoxicity following infection. These studies highlight the cytolytic function of antigen-specific CD8+ T cells elicited by M. tuberculosis infection and enable us to determine whether the cytotoxic function of CD8+ T cells is required for optimum pulmonary immunity to M. tuberculosis infection. Characterization of an Lck-Independent Pathway of T-Cell Activation Used by Bacterial Superantigens: Mechanistic and Therapeutic Implications on Autoimmunity.The current paradigm to explain activation of mature T cells following engagement of their T-cell antigen receptor (TCR) with peptide:MHC molecules relies on early activation of the src family kinase Lck. Lck phosphorylates tyrosine-based activation motifs on the subunits of the TCR complex, providing sites for recruitment and activation of the syk family kinase ZAP-70. ZAP-70 phosphorylates the transmembrane adapter LAT providing docking sites for the assembly of multimolecular complexes. These signalosomes trigger several signaling cascades that cause translocation / activation of transcription factors leading to changes in gene expression. Despite the requirement for Lck under most conditions, T cell activation can proceed in the absence of Lck under some circumstances. One of these involves the activation of T cells by superantigens (SAg), a type of stimuli implicated in the development of autoimmunity. Also, we and others have shown that TCR partial agonists with the capacity to induce T cell tolerance also use an Lck-independent TCR signaling pathway. We have dissected Lck-independent TCR signaling on human T cells upon stimulation with SAg. In contrast to Lck-dependent TCR signaling, early TCR signaling in the absence of Lck was characterized by lack of phosphorylation of ZAP-70, LAT and phospholipase (PLC) Cg-1. We found that SAg induced significant activation of the mitogen-activated protein kinases (MAPK) ERK-1/-2, Ca2+ influx, and translocation of NF-AT and NF-nB transcription factors in the lck-deficient T cells, leading to production of interleukin-2 (IL-2). The Lck-independent T cell response was blocked with protein kinase C (PKC) and MAPK inhibitors, but not by inhibitors of PI-3 kinase. In addition, we observed that IL-2 production by Lck-deficient T cells was enhanced by lithium chloride (LiCl) and minimally inhibited by pertussis toxin. Since LiCl stabilizes IP3 generation, which is involved in Ca2+ influx, we explored the involvement of alternative PLCs, in particular PLC-h. We found that inhibition of PLCh blocked Lck-independent activation of ERK-1/-2 suggesting that this enzyme is involved in TCR signaling. In conclusion, our data provide initial characterization of the Lckindependent TCR signaling induced by SAg. Since the activity of PLCh is regulated by heterotrimeric G-proteins, our data suggest that SAg-induced T cell activation involves a G protein-dependent pathway. These findings have implications to design inhibitors of T cell activation in the context of SAg-induced diseases. INTRODUCTION: The intensity of immune response as well as the predisposition to many diseases, including infectious illness, has been associated with specific HLA phenotypes.OBJECTIVE: To investigate the relationship between specific HLA phenotypes of patients and the predisposition to a chronic course of urogenital chlamydiosis in addition to in vitro production of IFN-g and interleukin (IL)-10 by peripheral blood mononuclear cells (PBMC).STUDY DESIGN: Seventy-eight patients with urogenital chlamydiosis were included in this study. HLA phenotype was determined by the standard Terasaki microlymphocytotoxic test utilizing a panel of anti-HLA-A and -B sera. Cytokine expression was assessed by commercial ELISA (Immunotec, France) of supernatants following in vitro culturing of PBMC with or without mitogen.RESULTS: Patients with chronic urogenital chlamydiosis were found to have significantly greater prevalence of the HLA antigens A10, B27, and B51. Individual analysis of HLA phenotypes demonstrated that IFN-g production was independent of the presence of these HLA risk-associated antigens. Conversely, significantly greater in vitro levels of IL-10 were observed in 83% of patients with the HLA risk-associated antigens for chronic urogenital chlamydiosis when compared to the study control subjects.CONCLUSIONS: HLA antigens A10, B27, and B51 can be considered as risk antigens associated with development of a chronic course of urogenital chlamydiosis. In vitro production of IL-10 by PBMC from patients with chronic urogenital chlamydiosis and these HLA risk-associated antigens is significantly greater than in control subjects. In contrast, IFN-g production is not dependent upon HLA phenotype. The chronic course of urogenital chlamydiosis in some patients may be associated with an immunosuppressive effect induced by interleukin-10.Sa2.64. Are Probiotics Essential Adjunctive Therapies for C difficile Enteritis? Recurrent C difficile gastroenteritis is increasing in both incidence and severity, especially in the young and elderly. Although the antibiotics flagyl and vancomycin are effective in ameliorating acute symptoms, both are often followed by recurrences after they are discontinued. Other antibiotics, which also kill normal flora in addition to the intended pathogen, also exacerbate or induce relapses in C difficile carriers who are otherwise asymptomatic. The following cases demonstrate the need to replace normal flora with a suitable probiotic, not only to hasten remission but to prevent recurrences and to eliminate the carrier state. Case 1) MG, age 85, was admitted for the third time from assisted care to Intensive Care for intravenous fluids and parenteral antibiotics. In 10 months she suffered 8 relapses, 6 requiring extended hospitalization and rehabilitation. Confined to isolation, she had severe abdominal pain, suicidal depression, thrush, and persistent diarrhea, only temporarily relieved by flagyl or vancomycin therapy. Stools were persistently positive for C difficile. As an alternative to long-term antibiotic therapy, the family investigated probiotics and asked her doctors to start it. They declined.After obtaining legal control of the patient, we discontinued both vancomycin and flagyl and started probiotics. Four enteric coated capsules (T Trio, Natren Co) containing 3 live organisms, L acidophilus, DDS-1 strain, B bifidum, Myeloff strain, and L bulgaricus were given q.i.d. Diarrhea recurred once when probiotics were inadvertently discontinued. C difficile was eliminated from the feces after 3 months and remains so with monthly testing for the past year.Case 2) BP, age 74, was admitted to a nursing home for a diabetic leg ulcer. He soon developed recurrent C difficile diarrhea treated with flagyl or vancomycin, which recurred whenever therapy was discontinued. For the next year, long after his leg ulcer had healed, he was in and out of the nursing home for retreatment of C diff. Finally probiotics were added, symptoms abated, and he was able to be discharged home. He also continues to take prophylactic probiotics, 1 or 2 caps/day and has remained well for the past year. Other similar cases, one a child, will be presented.We suggest that C difficile is an increasing problem not only because of immune deficiencies, but also because of the increase in use of antibiotics which kill the normal bowel commensal bacteria. These normal gut bacteria perform vital immunoregulatory functions and also close the tight junctions between endothelial cells which C difficile toxins open, thus allowing unregulated absorption of both toxins and allergens. The remarkable improvement of arthritis in case 1 fits the theory that certain antigen epitopes on gut bacteria may cross-react with synovial cells as the gut becomes more permeable. Probiotics should be considered essential for recurrent C difficile, either used alone or as adjunctive therapy. Post-trauma alterations in T-cell surface markers and functions are suggested to correlate with increased time to recovery and development of multiple organ dysfunction syndrome (MODS). Recently, dysfunctional co-stimulation, shifts in T-cell subset ratios and emergence of regulatory T-cells have all been suggested as contributors to immunological dysfunction. However, a definitive link between trauma patientsT T-cell immune alterations and clinical outcome is still undefined. Here, 31 trauma patients were divided into two groups based on the severity of their clinical course (average of the injury score and time to recovery from MODS; b30 versus N30). We assessed the surface expression of the co-stimulatory ligand-CD28 and the subset markers-CXCR3 (Th0/Th1), CCR5 (Th1) and CCR4 (Th2) to reflect shifts in T-cell populations or decreased activation. Regulatory T cell receptor levels-dual CD4CD25 and CTLA4, were also assessed to indicate any evidence of immunosuppression. These parameters were correlated to severity of clinical outcome b30 versus N30. Additionally, T cell intracellular cytokine (IL-2, IFNg, IL-4, GM-CSF and IL-10) levels in response to ex vivo PMA/Ionomycin stimulation were assessed. Patients (n = 31) from three different clinical centers were followed over 28 days post-injury (sampled at b12hrs and days 1,4,7,14,21 and 28) . Samples from normal subjects (n = 22) were simultaneously processed. Surface expression of CCR5 and CXCR3 were significantly decreased while CCR4 and dual CD4CD25 expression were significantly increased in T-cells from all trauma patients as compared to normal subjects. Similarly, stimulated intracellular cytokine (IL-2 and IFNg) levels were significantly reduced in T-cells from all trauma patients as compared to normal subjects. Some adaptive T cell post-injury alterations may occur in all patients while other selective aberrant alterations may be contributing to clinical pathology. Consequently, we compared alterations in these immune parameters between the patientsT groups with the most severe (N30) versus less severely (b30) clinical courses. Only IFNg and CXCR3 (pb0.05 at 14 days post-injury) expression were significantly reduced in patients with a poor clinical course, when compared to the less severely injured subjects. However, there was no significant difference in CCR5 or IL-2 between the two patient groups. These data suggest a selective defect in the Th1 populations from the severely injured patients, rather than a simple expansion of their Th2 cells. Such selective Th1 defects in severely injured patients could contribute to their increased clinical pathology. To determine the capacity of immune-based therapies (IBT) to boost HIV-specific immunity, a randomized, controlled, phase II trial tested IM ALVAC (vCP1452) F daily, low-dose SQ interleukin-2 (IL2) (1. Analyzed according to IBT regimens, the highest proportion of Rs was observed in the vaccine groups: placebo = 33%; vaccine = 78%; placebo + IL2 = 33%; vaccine + IL2 = 50%. These data indicate that a DTI is a valid clinical trial design to test IBTs, & support the notion that therapeutic immunization may improve the in vivo antiviral host response. An Automated Methodology for Evaluation of Dendritic Cells in Whole Blood. 1 1 Biomedical Research Division, Beckman Coulter Inc., Miami, FL, USA. Dendritic cells (DCs) are potent antigen presenting cells that play important roles in the induction of immune responses against microorganisms and tumors and are also vital players in the induction of tolerance and autoimmunity. Due to these unique features, DCs are very promising therapeutic tools being used to manipulate the immune system and form the basis of a number of ongoing clinical trials as biological adjuvants and modulators in cancer, infectious disease and autoimmunity. In order to further study the biology of these relatively rare DCs, as well as evaluate their efficacy in the various clinical trials, the easy identification and enumeration of these cells in whole blood, preferably in an automated and standardized format is pivotal.The current study was designed to develop an automated and standardized methodology for the phenotypic identification and enumeration of DCs in whole blood. The Beckman Coulter, Inc. 3color DC phenotyping reagents were used in conjunction with the COULTERR PrepPlus 2 automated pipetting instrument, the TQ-Prep automated lysing and fixing instrument, the CellPrep automated non-centrifugal washing instrument, and the Cytomics FC500 flow cytometer with totally automated instrument setup, data acquisition, and data analysis. Normal and abnormal whole blood samples were evaluated for identification and enumeration of DCs and results compared to the recommended validated manual techniques.The automated methodology enabled a more precise, and standardized bhands offQ identification and enumeration of DCs in whole blood. Signal to noise ratios were improved while maintaining similar cell recoveries compared to the manual methods in both normal and abnormal blood specimens. Therefore, the use of validated reagents, precise pipetting, automated preparation, automated cytometer setup, defined gating strategies, and automated data analysis and data reduction techniques enables a standardized phenotypic evaluation and enumeration of rare dendritic cell events in whole blood. Ozonetherapy and Quality of Life in HIV Positive Patients. Osmel Gaspar Guerra Segura, Barbara Elias-Calles Fernadez. Dr. A. Neto, Guantanamo, Guantanamo, Cuba; 2 Ozonetherapy Dept., Teach. Neto, Guantanamo, Guantanamo, Cuba.We have been done a prospective longitudinal study of positive HIV patients admited at the sanatory living in Guantanamo province in a period of two years. The sample includes 17 patients wich were divided into three aleatory groups. Th e first one was treated with Ozone, the second with a conventional treatment and the third with Ozone therapy-conventional treatment. Then we did an evaluation of some immunohematological and biochemist parameters and clinical success. At the begining, in 32 % of the patients the Eritro was high, lymphocytes subsets were within the normal limits and 69,2 % had an oxidative stress from moderate to severe and with a good evolution without opportunistic infections. Characteristics of Fas Ligand Expression by Human Cytomegalovirus Infection. 1 1 Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, Republic of Korea.The immune response is regulated not only by cell proliferation and differentiation, but also by apoptosis. Many viral infection disturb the normal regulation of apoptosis in the various cells. Although Fas-mediated apoptosis is one of the immune effector pathways leading to the elimination of virusinfected cells, viruses may escape immune surveillance by expressing Fas ligand (FasL) on the infected cells. Human cytomegalovirus (HCMV) infection was reported to induce FasL on retinal epithelial cells. This study was performed to find out the induction of FasL in various cell lines and to elucidate the characteristics of FasL expression in human fibroblasts upon HCMV infection. The constitutive FasL expression was found on T cell lines (Jurkat, H9 and CEM), but not on B cell line (Nalm6), osteogenic sarcoma cell lines (Saos-2, G292, HOS and MG63), astrocytoma cell ine (U373MG), lung cancer cell line (A549), human fetal lung fibroblasts (FLF), Epstein Barr virus-transformed lymphoid cell line (LCL) and human kidney cell line (293) by flow cytometry analysis. Upon HCMV infection with 2 multiplicity of infection, FasL expression was found only in Saos-2, FLF and LCL although some cell lines such as G292 and MG63 could be infected with HCMV. Time-course analysis of FasL expression with flow cytometry showed that FasL expression was found on the fibroblast surface immediately after HCMV infection. FasL expression was down-regulated at 12 hr and then up-regulated at 24 hr and thereafter, but down-regulated after 96 hr. FasL expression rate in FLF was increased dependent on the increasing titer of HCMV. Apoptotic death of Jurkat cells were measured by propidium iodide staining and flow cytometry for the detection of FasL expression on the cell surface of FLF after Jurkat cells were co-cultured with FLF infected by HCMV with various infectious doses. It was found that the apoptotic death rate of Jurkat cells on the HCMV-infected FLF was proportional to the infectious doses of HCMV to FLF. Relative changes of mRNA expression of FasL and HCMV immediate early (IE) and early proteins were measured by real time polymerase chain reaction in FLF after HCMV infection at sequential time points. HCMV infection up-regulated FasL mRNA in less than 6 hr but down-regulated to the basal level from 12 hr to 24 hr and then up-regulate FasL mRNA thereafter, but down-regulated after 96 hr. It was reported that only HCMV IE2 activated the promoter of FasL, but in this experiment HCMV IE1 and IE2 mRNA were increased and persisted continuously except in 72 hr, and HCMV UL44 mRNA were increased and persistent. These results mean that other factors than HCMV IE2 involve in the regulation of FasL expression. Conclusively the functionally active FasL could be induced in the restricted cell types by HCMV infection and would be regulated with many cellular and viral factors in addition to the earlier reported HCMV IE2. IC41 is a vaccine consisting of five synthetic peptides harboring relevant CD4+ and CD8+ T-cell epitopes of Hepatitis C Virus (HCV) and the synthetic Th1/Tc1 adjuvant Poly-L-Arginine. In recent multicenter studies this novel vaccine was investigated in more than 200 subjects. To monitor immunogenicity, PBMC were cryo-preserved and T-cell responses were assessed by interferongamma ELIspot and lympho-proliferation. In addition, a highly sensitive five-color HLA class I tetramer-binding assay was developed to quantitate and phenotype HCV-specific CD8+ cytotoxic T cells. A HLA-A*0201-transgenic mouse reference standard was established as long-term quality control of the custommade HCV tetramers used in these clinical longitudinal studies. Here we present the detailed analysis of a well known HCV NS3derived HLA-A2 epitope part of IC41. This included staining for a panel of different surface markers (CD45RA, CCR7, CD27, CD28, CD38, and HLA-DR), intracellular interferon gamma and perforin. The evolution of the HCV-specific T-cell response was compared to recall responses against CMV. Moreover, CD8+ T cell responses against the NS3 epitope seen in tetramer or ELIspot analyses were correlated with proliferative CD4+ T cell responses. Most importantly, several chronic HCV patients with T cell responses against this epitope showed a concomitant yet transient decline in HCV RNA. In summary, these data indicate that therapeutic vaccination with IC41 can induce cellular immunity against HCV, and results from these studies provide first insights in the mode of action of IC41 on human T lymphocytes.Sa2.71. Objectives: Fibrinogen-like protein 2/fibroleukin (fgl2) plays a pivotal role in the pathogenesis of both experimental and human fulminant viral hepatiits. We have previously reported that the nucleocapsid protein of murine hepatitis virus 3 which causes massive hepatocellular necrosis in Balb/cJ mice induces transcription of mfgl2. The aim of this study was to investigate the key factor(s) of hepatitis B virus (HBV) that induces transcription of human fgl2 (hfgl2) and the cis-acting elements involved.Methods: three eukaryotic expressing plasmids of HBV structure genes encoding C, X and S proteins respectively were constructed. Expression of these plasmids in eukaryotic cells were detected by immunohistochemistry and Western blot. A series of 5V truncated promoter of hfgl2 gene were subcloned into the luciferase report vector pGL2-basic to form the promoter-report constructs. Chinese hamster ovary cells and HepG2 cells were cotransfected with constructs expressing HBV X, C and S protein respectively and a hfgl2 promoter construct upstream of the luciferase report gene.Results: HBV X protein and C protein but not S protein activates the transcription of hfgl2 gene, showing a an average of 6-fold and 5.4-fold increasing in relative luciferase activity. Further more, series deletion array of hfgl2 gene promoter show that a strong regulatory domain from-816 to -468 (relative to the transcription start site) is responsible for the regulation of hfgl2 gene transcription in response to HBV proteins.Conclusion: These results indicate that HBx and HBc proteins are the viral factors that involved in the hfgl2 expression, and the promoter region of-816 to-468 may contain the cis-elements in response to viral proteins C and X. This study provides new insights in understanding the interaction between virus and host gene expression and its contribution to the pathogenesis of viral fulminant hepatitis. This work was supported by the National Science Fund Regulatory CD4+ T cells (Tregs) are critical for control of protective immune responses to foreign organisms. We hypothesized that BCG vaccination of the newborn induces specific Tregs, and that a dynamic equilibrium between specific regulatory and conventional immunity exists.We incubated whole blood from 36 10-week old South African infants, routinely vaccinated with BCG at birth, with BCG and determined mRNA expression of the Treg-specific marker Foxp3 (real time RT-PCR). A median 19% (range of 0-650%) upregulation of Foxp3 strongly suggested presence of specific Tregs.Tregs may suppress conventional immunity through either TGF-h, or IL-10, or CTLA-4. We therefore incubated whole blood of vaccinated infants with BCG in the presence of neutralizing antibodies against these molecules, and showed a median 11% to 25% increase in soluble IFN-g production (ELISA), suggesting that these Treg effector molecules do indeed control conventional BCG-induced immunity.Because these effector molecules may be made by cells other than Tregs, we correlated BCG-induced mRNA expression of these molecules with that of FoxP3. This suggested that TGF-h may be the effector molecule of mycobacteria-specific Tregs.To determine whether IL-10-producing Tregs (Tr1 cells) were present, we measured BCG-induced CD4+ T cell-specific expression of this cytokine (flow cytometry). Low frequencies of CD4+ T cells made IL-10 (median 0.01%). However, individual cells were either IL-10+ or IFN-g+, but not double+, suggesting presence of both specific Tr1 and specific conventional CD4+ T cell subsets. )Finally, we found that BCG-induced IFN-g mRNA expression correlated with Foxp3 expression (r2=, P b 0.001), suggesting a dynamic equilibrium between specific regulatory and conventional immunity.We conclude that newborn vaccination with BCG does induce specific Tregs, which may control conventional effector/memory T cell immunity. INTRODUCTION The monocyte locomotion inhibitory factor (MLIF), an antiinflammatory pentapeptide (Met-Gln-Cys-Asn-Ser) produced by Entamoeba histolytica, inhibits the in vitro and the in vivo locomotion of human monocytes (PBMN), without affecting that of neutrophils or eosinophils. MLIF also virtually cancels the production of reactive oxygen and nitrogen intermediates (ROI, RNI) in PBMN and nPMN, and again spares eosinophils from this effect. MLIF also inhibits delayed hypersensitivity skin reactions to 1-chloro-2-4dinitrobenzene in guinea pigs and gerbils. All this may contribute to the regeneration without scarring of the affected organs (i.e. liver, skin) found on recovery after treatment. OBJETIVE To determine the effect of MLIF on the profile of inflammation/ woundhealing gene expression in the human cell line MRC-5, using a microarray device. MATERIAL AND METHODS A 96% pure construct of MLIF and the human cell line MRC-5 were employed. The cell line MRC-5 was expanded in MEM containing 10% bovine fetal serum until reaching the adequate cell count. Cells were adjusted at 5Â10 6 cells/5 ml and were placed in 6-well plates, incubating them for 24h at 378C with 5% CO 2 in: PMA-Ionomicine (10ng/10 6 cells/ml, 0.5Ag/10 6 cells/ml), or PMA-Ionomicine+MLIF (10ng/10 6 cells/ml, 0.5Ag/10 6 cells/ml, 50Ag/ 10 6 cells/ml). Controls consisted of cells incubated without any stimulus or cells exposed to 50Ag/10 6 cells/ml MLIF alone. Cells were treated and lysed in 1ml of TRIZOLR. Total RNA was isolated following the manufactureŕs instructions and DNA was removed with DNAase I. RNA integrity was confirmed by agarose gel electrophoresis.The synthesis of labelled cDNA with 33 P was started from total RNA (Human Cytokine Labelling PrimersR) and purified in Sephadex G-25R columns. The cDNA was hybridized in gene microarray membranes (Panorama Human Cytokine Gene ArraysR) Membrane were exposed using BioMax MR X-ray filmR. Spots were quantified by digital analysis. Average values of housekeeping genes and negative controls were used to adjust values of the genes in the membrane. We used 0.999 % confiability and a Z value of 3.090. bInvariantQ TCR-a+, non-invariant diverse ah+, and gy+ CD1d-restricted T cells are subsets of dNKTT cells with distinct locations and functions. CD1d-restricted T cells can rapidly produce large amounts of anti-&/or pro-inflammatory cytokines and chemokines depending on microenvironment and stimulus. Systemic stimulation of invariant NKT cells leads to transient protection against numerous diseases through indirect activation of various antigen presenting cells (APC), B and T cells, all of which can express CD1d, as well as NK cells. CD1d-restricted T cell activation can rapidly stimulate APC pro-inflammatory IL-12 production. In vivo, both invariant NKT cell activation and CD1d ligation could protect against acute picornavirus infection, suggesting re-examination of results involving CD1d bblocking.Q CD1d-restricted T cells are also essential for optimal physiological response against picornavirus through augmentation of IL-12 production and NK cell activation. A case report further supports an antiviral role for human invariant NKT cells. However, excessive activation of local CD1d-restricted T cells can lead (directly and indirectly) to tissue damage. High levels of IFN-g producing Th1 resident hepatic CD1d-restricted T cells may protect against acute infection, yet contribute to pathology during chronic infections such as hepatitis C through amplified and modified Th1/ Th2 responses to tissue CD1d. Analogous results were found in model viral myocarditis. Invariant NKT cells are also essential for optimal model anti-tumor responses against spontaneous cancer, metastases, and tumor vaccines. Cancer progression correlates with failure of patient invariant NKT cells to activate protective antitumor responses. This defective response can be corrected in vitro and a phase 1 trial has been designed. Finally, the presence of NK cell markers is not essential for function. Therefore, CD1drestricted T cells are diverse multi-functional cells at the innateadaptive immune response interface. Therapeutic approaches exploiting distinct CD1d-restricted T cell populations are being explored, for example in graft-versus-tumor responses and graftversus-host disease suppression. Objectives: To establish a substitutive murine model of severe acute respiratory syndrome (SARS) with murine hepatitis virus type 3 (MHV-3) and to investigate the interaction between SARS virus and host immune response, mfgl2 prothrombinase gene as a representative of host genes.Methods: The Balb/cJ mice were infected with 100PFU of MHV-3 via trachea and Balb/cJ mice injected with sterilized saline were served as control. Survival rate and pathological features in organs were observed. Virus titers in different organs were determined on monolayer of L2 cells by a standard plaque assay. Virus distribution and cellular localization were studied by in situ hybridization. As a proinflammatory gene, mfgl2 expressions were measured in the lung by in situ hybridization and immunohistochemistry to investigate the role of mfgl2 in the pathogenesis of the disease.Results: Mice infected with MHV-3 via trachea developed typical interstitial pneumonia with pathological characteristics of leukocyte infiltration, hyaline membranes formation, exudation of fibrin fluid within the lung, presence of vascular thrombosis in the pulmonary vessels and died within 5 days, while all mice in control group survived with no inflammation in lung. Moderate histological changes were also found in liver and spleen but mild in other organs examined. MHV-3 particles were identified in the cytoplasm of alveolar epithelia, infiltrating cells in the lung, serous gland epithelium of the trachea/bronchus, cells (lymphocytes and others) in the periphery of the germinal centers in the spleen, hepatocytes, brain neurons, epithelia of the distal renal tubules, epithelium of small intestine, myocardium cells. mfgl2 expression was evidenced in lymphocytes and mononuclear inflammatory infiltrates within stroma and in terminal and respiratory bronchioles associated with fibrin deposition and micro-vascular thrombosis.Conclusions: The pathological changes in this substitutive SARS murine model mimic the characteristics of SARS in human. In addition to the physical damage of virus replication in lung and immune organs including spleen, liver, the up-regulation of novel gene mfgl2 in lung in association with fibrin deposition and microvascular thrombosis may play a vital role in the development of Recognition of microbial infection and initiation of host defense responses is controlled by multiple mechanisms. B cells provide an important link between the innate and adaptive branches of the immune system. However, direct involvement of B cells in imparting potentially protective responses is unclear. We here focused the relevance of naive B cells via TLR9/RP105 to innate immunity by secreting essentially germline encoded polyreactive antibodies. The proliferation of CD27 + memory B cells was higher than that of CD27naive B cells by CpG DNA stimulation as reported. The proliferation of naive B cells was extremely higher than that of memory B cells by CpG DNA and anti-RP105 mAb stimulation. The expansion and cell size were dramatically increased by combined CpG DNA and RP105 signaling. The life span of naive B cells was prolonged by anti-RP105 MAb and CpG DNA plus anti-RP105 MAb. Total immunoglobulin and Staphylococcus Pneumoniae specific IgG/ IgM production by adult naive and cord blood B cells ware recognized in the presence of CpG DNA/anti-RP105 mAb. Interestingly, IL-21 enhanced the production of Staphylococcus Pneumoniae specific IgG/IgM. However, the differentiation of B cell to plasma cells was not affected by anti-Rp105 mAb stimulation. Naive B cell activation via TlR9/RP105 pathway may play a beneficial role through profound their expansion and generation of antigen specific low affinity antibodies. This innate immune system by naive B cells may detect infections and trigger antimicrobial host defense responses. RESUMEN Introducción: Las células con funció n inmuno-reguladora más ampliamente caracterizadas son los Linfocitos T CD4+ CD25 bright cuyo papel en la regulació n de enfermedades inflamatorias y autoinmunes es preponderante. Por otro lado la tuberculosis es una enfermedad causada por el bacilo Micobacterum tuberculosis y se caracteriza por cursar con daño tisular severo, en el pulmó n, debido principalmente a la propia respuesta inmune del enfermo por lo que posiblemente los mecanismos de regulació n de esta respuesta inmune quizás estén alterados. En este estudio analizamos el numero de células inmuno-reguladoras en sangre periférica de pacientes con tuberculosis en comparació n con sujetos sanos, así como la expresió n del gen GITR el cual se ha reportado que esta presente en estas células y que abate su funció n supresora.Objetivo: En ratones las células T reguladoras CD4+ CD25+ tienen un papel preponderante en la prevenció n de fenó menos de autoinmunidad. En este trabajo investigamos la presencia y el fenotipo de estas células en la sangre periférica de individuos con tuberculosis pulmonar activa y en sujetos sanos.Métodos: Se estudiaron doce pacientes con tuberculosis y doce sujetos sanos. Se analizó el numero de las diferentes subpoblaciones de linfocitos T CD4+ en sangre periférica por citometria de flujo, así como la expresió n de GITR por RT-PCR en células mononucleares de sangre periférica.Resultados: Los niveles de los diferentes tipos celulares tanto en pacientes como en los sujetos control en la sangre periférica tienen un rango de valores muy amplio, no observándose diferencia significativa entre ambos grupos a excepció n de las células productoras de IL-10 que en los pacientes es mas grande comparado con los controles. Conclusiones: Para las células Treg., las que producen TGFb y las CTLA-4+ nuestros resultados muestran valores similares tanto en pacientes como en sujetos sanos que nos indica que estas células no se ven afectadas con la presencia de la enfermedad solo las que son IL-10+ muestran valores mas elevados en el grupo de los pacientes aunque este estudio se llevo a cabo en sangre periférica en donde el numero de estas células es muy pequeño con lo que pensamos que seria de gran importancia el desarrollar el mismo estudio pero directamente en el sitio de la lesión. Direct Epitope Discovery for Yellow Fever Virus Asibi-17D. A. Collymore-Slovak, 1 A. Gilb, 1 C. Roberts, 1 M. Jones, 1 W. H. Hildebrand. 1 1 Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.Major histocompatibility complex (MHC) class I molecules bind intracellular peptides for presentation to cytotoxic T lymphocytes (CTL). CTL respond to foreign peptides presented by the class I of virus-infected and cancerous cells. Identification of peptides presented by class I molecules during a yellow fever virus (YFV) infection is important for understanding in vivo immume responses to infection and for comparing these responses to those arising from vaccination with Yellow Fever-Asibi [17D] . YFV belongs to the family Flaviviridae and is a positive-sense, singlestranded, encapsulated RNA virus. The virus enters cells by receptor-mediated endocytosis, viral RNA synthesis occurs in the cell cytoplasm, and viral protein synthesis proceeds in the endoplasmic reticulum. Virions mature in the endoplasmic reticulum and are released through the cellTs plasma membrane by exocytic fusion. YFV is spread to a human host when the saliva of an infected female mosquito mixes with capillary blood during a meal. The virus then infects vascular endothelial cells and is able to spread to the reticuloendothelial system where it replicates and secondary viremia may occur causing the immune system to be overwhelmed. To understand which peptide epitopes distinguish the class I MHC of YFV-infected cells, we selected the U937 human macrophage cell line for characterization. We transfected HLA-A*0201 and B*0702 into the U937 cell line and then infected the cell line with YFV strain 17D. U937 cells secreting either A*0201 or B*0702 are cultured in a hollow fiber bioreator unit, the class I HLA is harvested from both infected and uninfected cells, and the peptide epitopes unique to YFV infected cells identified by comparative mass spectrometric mapping and MSMS sequencing of peptides unique to YFV infected cells. Identification of MHC class I epitopes unique to YFV infected cells will be useful for understanding to which of the epitopes available on infected cells does the YFV vaccine elicit, or fail to elicit an immune response.Sa2.79. Clonal Organization of T Cell Memory: Paradigm of bFocused DiversityQ Contributes to Repertoire Flexibility. Yuri N. Naumov, 1 Shalyn C. Clute, 1 Elena N. Naumova, 2 Jack Gorski, 3 The human memory CD8 T cell receptor (TCR) repertoire specific to the influenza A virus, HLA-A2.1-restricted M1 58-66 epitope was analyzed. b-TCR clonotypic sequences of tetramerselected cells or cells from the bulk cultures revealed a clonally diverse population of responding T cells. These cells used BV17 almost exclusively and focused on a similar CDR3b structure, a paradigm we refer to as bfocused diversityQ. This was observed in four different HLA-A2.1 individuals. We determined a strong clonal selection based on a-TCR usage as all expressed a-TCRs originated from AJ42 and multiple AV (AV27, AV8.1, AV 8.6 and others) families. Furthermore, there was a CDR3a motif identified utilizing AGA(G n )GG or similar amino acid sequences. Some of these CDR3a only differed by the length of the run of Gly residues. Thus, the pattern of TCR repertoire in the context of bfocused diversityQ extends to both of the TCR chains. This complex repertoire structure was highly resistant:-not only was it stable over time, but persisted under condition of highly variable antigen stimulations in vitro. These results suggest that the ab-TCR repertoire flexibility in the context of bfocused diversityQ might allow antigen-specific immune responses to accommodate extremes of antigen challenges. T lymphocytes recognize antigens presented by antigen presenting cells (APC). Peptides are presented by the MHC system, with endogenous peptides presented by MHC class I and exogenous peptides presented by MHC class II. In contrast to the MHC system, CD1 molecules survey different intracellular compartments for lipid antigens which are presented at the APC surface to lipid-reactive T cells. Recently, saposins were shown to mediate an important step in presentation by transferring lipid antigens from lysosomal lipid bilayers to CD1 molecules. However, the mechanisms of exogenous lipid antigen delivery to CD1 antigen loading compartments are not known. Serum lipoproteins are the primary mediators of extracellular lipid transport for metabolic needs, however their role in lipid antigen presentation has not been investigated. Here, we define the pathway mediating exogenous lipid antigen delivery by apolipoprotein E (apoE) to achieve antigen presentation by CD1. In human serum, lipid antigens sequester in the very low density lipoprotein (VLDL) fraction with antigenicity being dependent upon apoEmediated uptake into dendritic cells. In addition, we have found that dendritic cells themselves secrete apoE, which can then efficiently capture lipid antigens. ApoE-lipid antigen complexes are internalized by receptor mediated uptake resulting in rapid and efficient antigen delivery to endosomal lipid antigen loading compartments enabling CD1 restricted T cell activation. The immune system has thus co-opted an important component of lipid metabolism for the presentation of lipid antigens. Major histocompatibility complex (MHC) proteins are encoded by highly polymorphic genes and play a crucial role in immunity. The polymorphisms that distinguish MHC molecules are predominantly positioned to modify peptide binding. However, recent studies seem to indicate that not all genetically diverse MHC molecules are functionally different. As such, classification of MHC molecules into functional supertypes on the basis of overlapping peptide-binding specificities has become an important issue, with direct implications for the development of epitopebased vaccines with wide population coverage. Unfortunately, direct experimental validation of multiple members of these supertypes has been tremendously difficult because of lack of high quality human leukocyte antigen (HLA) molecules. In this study, we describe a direct biochemical approach for classifying HLA molecules into supertypes using recombinant soluble HLA (sHLA) proteins. A set of 53 single-specificity sHLA receptors was screened for their binding capacity towards a series of unrelated fluorescent-labeled peptide ligands. During the process, each peptide candidate was incubated with activated sHLA, and the peptide/HLA interaction was monitored over time. Data analysis showed various degrees of overlapping peptide binding capacities among the alleles tested, reflecting the ability of MHC class I alleles with genetically more or less distinct peptide binding sites to share the binding of identical peptides. As a result of this study, we were not only able to better define supertype classification but also to include additional alleles not currently characterized. Taken together, this novel HLA screening procedure represents a versatile tool for supertype-binding analysis and will have profound use in the understanding of antigenic peptide selection, degeneration and discrimination during T-cell mediated immune responses. A complete knowledge of this phenomenon finds utility in epitope design for the development of HLA based vaccines and immunotherapeutics. Development and Validation of Fluorescence Polarization-Based Competitive Peptide Binding Assays-New Screening Tools for Epitope Discovery. Various approaches are currently proposed to successfully develop therapies for the prevention and treatment of infectious diseases and cancer. One of the most promising approaches is the development of vaccines that elicit cytotoxic T lymphocyte (CTL) responses. In order to facilitate CTL-specific epitope discovery and validation, we have developed a state-of-the-art biochemical HLA class I peptide-binding assay including A*0101, A*0201, A*0301, A*1102, A*2401, A*2902, A*6801, A*6901, B*0702, B*0801, B*1501, B*2705, B*3505, B*4001, B*4402, and Cw*0801. The technique is based on competition and uses FITC-labeled class I binding reference peptides in combination with highly purified, recombinant soluble HLA class I molecules to quantitatively measure the binding capacity of non-labeled peptide candidates. The assay shows excellent reproducibility and sensitivity with dynamic ranges between 170 to 240 mP. Optimal loading of synthetic peptides into fully assembled soluble HLA-A*0201 complexes is enabled by thermal destabilization at 538C for 15 minutes demonstrating that efficient peptide exchange does not require the removal of endogenous peptides from the reaction environment. Multiple assay-specific parameters have been determined showing that the binding of the selected fluorescent peptides to soluble HLA is effective with many of the molecules (21-37%) binding exogenously added peptides. To further explore the specificity of binding and to demonstrate the immunologic relevance of the FP-based competition assay, a panel of HLA A*0201-restricted and naturally processed peptides have been tested for binding to sHLA-A*0201. In this validation approach, a panel of 15 peptides was selected to represent binders that reflect several orders of magnitude of A*0201 binding affinity. Obtained IC 50 values were directly compared to the values published from direct cell-free I 125 -radioligand assays and cellular based fluorescence assay systems. Results proved to be in excellent agreement with each other also allowing the formulation of an FPbased category system, where peptides with an FP-based IC 50 value of 5 AM and lower were considered high affinity binding, 5-50 AM IC 50 values were considered medium-affinity binding, and 50 -1,000 AM IC 50 values were judged as low-affinity binding. With the ability to exactly determine molecular affinity parameters, our FP-based HLA competition assays are of prime importance for the development of immunomodulating compounds. Built into a high-throughput screening platform, this new assay approach will bridge current screening gaps and considerably shorten the route to clinical development considering the myriad of possible peptide sequences, coupled with the genetic variability of MHC molecules among individuals. Anthrax Toxins Impair Pulmonary Dendritic Cell Functions: Implications in Pathogenesis. A. Cleret, 1 A. Quesnel-Hellmann, 1 J. Mathieu, 1 D. Vidal, 1 J. -N. Tournier. 1 1 Immunology Unit, CRSSA, La Tronche, France.Anthrax is a life-threatening disease of present and future concern, especially since it was used as a bioterrorist weapon in 2001. To investigate the role of toxins in the physiopathology of inhalational anthrax, we evaluated the functions of murine lung dendritic cells (LDC) upon infection with B. anthracis strains secreting LT, ET, both toxins, or with a non toxinogenic strain. Briefly, isolation of LDC from BALB/c mice was performed after digestion by collagenase and DNase followed by positive selection with CD11c microbeads. We purified three cell populations gated on CD11c/CD11b expression: (i) a CD11c high /CD11b low ; (ii) a CD11c int /CD11b int and a (iii) CD11c low /CD11b high population.Upon infection with LT expressing strains, we observed a down-regulation of CD86 and MHC class II expression on CD11c int cells. LDC infected with a non toxinogenic strain induced the secretion of TNF-alpha, IL-10, IL-6 but a low level IL-12p70. LT producing strains selectively inhibited the production of TNF-alpha, IL-10 and IL-6, whereas ET producing strains inhibited TNF-alpha, did not affect IL-10 and increased significantly IL-6 production, as compared to a non toxinogenic strain. These results suggest that anthrax toxins produced during infection impairs LDC functions and suppress the innate immune response. This may represent a new strategy developed by B. anthracis to escape the host response and spread through the alveolar wall. Rheumatic fever is an inflammatory autoimmune condition following 3% of non treated group A streptococci pharyngitis of certain M serotype strains. Heart valves are permanently damaged by inflammation leading to heart valve replacement surgery. In addition to a cellular heart valve attack, anti-basal ganglia autoantibodies arising later in the autoimmune process target caudate and subthalamic nuclei antigens, being responsible for the neurologic symptoms of SydenhamTs chorea. Antibodies targeting both heart antigens and M proteins were found in blood of affected patients. M protein and cardiac valve derived proteins were shown to be recognized by T cell clones obtained from lymphocytes infiltrated in rheumatic patient cardiac valves (Guilherme et. Therefore, antigenic mimicry between streptococcal M protein epitopes and heart components has been proposed as the triggering factor leading to the heart autoimmune attack. The understanding of the disease process and therapeutic advances has been hampered by the lack of an adequate animal model for the disease. We have injected Lewis rats with streptococcus recombinant M1 protein 500ug on day 0 followed by 500ug boost on day 7 and sacrifice on day 21, in order to reproduce a recently described animal model of rheumatic fever (Quinn, A. et al, 2001, Infect.Immun. Rat hearts were subjected to histopathological analyzes. Spleen and lymph node lymphocyte cells, as well as sera, were harvested and probed against ABC domains of the M1 complete extracellular protein, AB domains (N-terminus), or C domain (C-terminus) and myosin or control proteins. Rat-immunized cells were used for FACS analysis to study their phenotypic profile and cytokine production. We have obtained specific lymphoproliferative and humoral immune responses against M1 ABC, AB and C domains of the S.pyogenes M1 protein. The proliferation index reached above 10, and the antibody titers were significantly higher than in controls, up to the 12.800 fold dilution, but we could not detect any proliferative nor humoral response against myosin. The anathomopathological evaluation of rat hearts displayed presence of lymphomononuclear infiltration and signs of lesions suggestive of of Rheumatic Fever in some of the immunized animals. We are currently analyzing different immunization protocols in order to reproduce the disease in Lewis rats. We believe that the definition of the M protein minimal epitope(s) responsible for RF will certainly contribute to better understanding of the disease process. The majority of the studies done mainly in murines show the resulting alterations from Zinc deficients and the stimulus or the protection induced by the metal in some cell cultures, however, there are evidences that demonstrate that Zinc supplementation in vitro or in vivo can cause disturbs in the immune system.Our study has as objective to establish which is the influence of Zinc in the maturity and viability of different cellular lines (U937, human monocytes and mice bone marrow macrophages), as well as the effect that and identical stimulus (proportioned by Zinc) can generate in the same cellular line but from different origin.The monocytes were obtained from vein blood from healthy individuals through the Boyqm method. The bone marrow cells were obtained from the femur of BALB/c mice, 6 to 8 weeks old, and the U-937 cells from aliquots maintained in culture.5Â10 6 cell/well were added in sterile plastic micro plates, then it was added the medium required for each cellular line with or without the stimulante factor required for its differentiation (supernatant of L 929 cells with the granulocyte macrophage colony stimulated factor for the bone marrow cells of mouse and PMA for the human monocyte and U-937 cells) without Zinc or with different dose of metal (0.05-1.0 mM). They were in culture for 11 days at 378C with 5% of CO 2 . Microscopic observation were done at 3,9 and 11 days, for the morphologic analysis and cellular differentiation; the viability and total recount of cells were determinated on days 6 and 11.The viability of cells of bone marrow of mouse, mononuclear phagocytes and U-937 cells incubated with 0.05 and 0.1 mM of Zinc was similar to the controls without Zinc (90%). With 1.0 mM of metal, viability decreased considerably (P b 0.0006), 50% of the mouse bone marrow cells and human MP, 80% in U-937 cells. The number of cells increased and there was no cellular differentiation for the morphologic characteristics observed at the beginning of the culture (small and round cells) were not modified during the stated lapse of time (11 days). Deleterious effect induced with this dose of Zinc was observed in the three cellular lines.Variations in number, size, morphology and viability of the cells observed with the different concentrations of the metal, and/or with the same doses at different times suggest that the effect exerted by Zinc depends not only of the used dose but also of the period of its interaction with cells. At present, there are only a few effective therapeutic approaches for the treatment of septic patients and mortality rates still remain high. In the present study we elucidated the potential role of the recently discovered heterodimeric cytokine in the pathogenesis of septic shock or acute polymicrobial sepsis.In a first series of experiments, we injected mice deficient for the EBI3 subunit of IL-27 or WT controls intraperitoneally with 40 mg/kg E. coli LPS. As a result, EBI3 deficient mice survived significantly longer than WT mice following endotoxin administration. Furthermore, in the cecal ligation and puncture model (CLP) of polymicrobial sepsis EBI3 deficient mice displayed improved long-term survival (45 %) compared to control mice, which almost all died within 72 h post CLP.After systemic administration of recombinant IL-27 into EBI3 KO mice immediately after CLP these mice died within 2 days suggesting a pathogenic role of IL-27 in the immune pathology of the disease. Mini-laparoscopic analysis demonstrated that IL-27-EBI3 deficient mice had a significantly reduced inflammatory response in the peritoneal cavity compared to wildtype controls after CLP.We next investigated cytokine levels in serum and peritoneal lavage at different time-points after induction of septic peritonitis. As a result IL-27-EBI3 KO mice showed a markedly attenuated proinflammatory cytokine response as indicated by decreased expression of IL-6 in both blood and peritoneal lavage, whereas bacterial concentrations in blood were similar to those in control mice. Subsequently, we analyzed the kinetics of IL-27 expression after CLP by quantitative Realtime-PCR. The expression of both IL-27 subunits was strongly induced in liver, spleen, lung and peritoneum 4, 6 and 8 h post CLP. Interestingly, peritoneal macrophages were the major source for IL-27 expression. To determine the cells/tissues that are targets for the biological functions of IL-27 during sepsis, we analyzed the expression of the IL-27 receptor chains WSX-1 and gp130 in cells, which have been implicated in the pathogenesis of the disease. We found out, that Mast cells and neutrophil granulocytes co-expressed both receptor chains suggesting that IL-27 can induce the release of proinflammatory mediators in these cells. CONCLUSION: Our data implicate that IL-27 has a critical role in the early pathogenesis of septic peritonitis and may be a potential new therapeutic target for this disease. The structural and functional integrity of the BBB is altered by various diseases of the CNS such as HIV AIDS Dementia (HAD). HIV-1 enters the CNS via the transmigration of infected monocytes across the BBB. Drug abuse in HAD patients leads to greater fluidity of cellular membranes which, increases the permeability of the BBB to immunocompetent cells. The efflux transporter Pglycoprotein (P-gp) is an important component of the BBB and an HIV-1 induced increase in P-glycoprotein expression could be one of the mechanisms for reduced intracellular accumulation and resistance to anti-retroviral agents resulting in the progression of HAD. Morphine is transported by P-gp therefore the BBB permeability of morphine may be altered based on the level of P-gp expression in the endothelial cells of the BBB. We hypothesize that one of the mechanism by which morphine increases the transmigration of HIV-1 infected monocytes across the BBB is via the modulation of P-gp expression by the brain microvascular endothelial cells (BMVEC) that constitute the BBB. Using an invitro BBB model, we studied the effect of morphine (10 À7 to 10 À11 M) on transmigration of HIV-1 infected monocytes and on Pgp gene and protein expression by BMVEC. The monocytes were infected with HIV-1 IIIB strain and were used in transmigration assays. P-gp gene expression was quantitated using real time, quantitative PCR while protein levels were measured using western blot analysis. Our results show that morphine significantly increased the transendothelial migration of uninfected monocytes by 39% (P b 0.01) as compared to control and it further enhanced transmigration of HIV-1 infected monocytes by an additional 23%. Morphine at 10 À7 and 10 À9 M concentration alone and in combination with HIV-1 tat (10-100ng/ml) significantly increased P-gp gene and protein expression in BMVEC. Increased P-gp expression in BMVEC results in enhanced BBB permeability to HIV-1 infected monocytes contributing to the neuropathogenesis of HIV-1 in drug abusers. Rheumatic fever (RF) is a post-infectious autoimmune disease prevalent in over 20 million children, mostly in developing countries. It is triggered by Streptococcus pyogenes and affects susceptible individuals following a non-treated throat infection. Thirty to 45% of patients develop carditis, the most serious disease manifestation leading to severe and permanent valvular lesions and to the development of chronic rheumatic heart disease (RHD). We have been studying the mechanisms leading to pathological autoimmunity in rheumatic fever and RHD for the last 15 years. Our studies have contributed to a better understanding the cellular and molecular basis of RHD, opening a gateway for the development of a vaccine for a post-infectious autoimmune disease. We studied the Cterminal region of the M protein, in which we have identified some potentially protective epitopes, recognized by both T and B lymphocytes from more than 500 healthy individuals and RF/ RHD patients. In order to verify potentially autoimmune epitopes we analyzed 21 overlapping C-terminal peptides (20 mers each differing by only two amino acid residues) and 9 N-terminal peptides previously described as heart-tissue cross-reactive by proliferation and cytokines secretion assays using 09 heart-infiltrating T cell lines (HIL) obtained from rheumatic heart disease (RHD) patients. Our results showed that 5 out of 9 HIL were able to recognize 7 Cterminal peptides. Among these peptides, four Our results indicate that peptides differing by only a few amino acids are capable of generating divergent immune responses, which could be the key issue in defining vaccine epitopes. Intralesional T Cell Lines Obtained from Sa2.89. Is There Any Relation between Lymphocyte Subsets, NK Activity and Infection in Beta Thalassemia Patients. In h-Thalassemia is multiple transfusion therapy is used to maintain nearly normal hemoglobin levels, and partially suppresses the increased, but ineffective erythropoiesis. Unfortunately, transfusion is associated with alloimmunization, risk of exposure to infectious pathogens and accumulation of iron. There is some contaversy in relation between immunological status, transfusion therapy regimen and frequencuy of infection and to date it is not quite clear why these patients are suseptible to infectin.In this study lymphocyte immunophenotyping for CD3+ (Tcells), CD3+/CD4+ (T-helper cells), CD3+/CD8+ (T-cytolytic), CD3+,CD19+ (B-cells) and CD3-/CD16+,CD56+ (Natural killer cells) was detected in the whole blood of 30 beta thalassemic patients using a three -color flow cytometric technique, Nnatural Killer cell activity was a also determined, with flow cytometric assay for NK cytotoxicity using PKH2-labled K562 target cells. Serum ferritin was measured by RIA as the iron status. We recruited 30 healthy children of comparable age that had never gone under blood transfusion. Information about the first date of transfusion, history of infection and age of adminestring chalation therapy were obtained by interview, archive files and related physician.Results showed an increased CD19+ lymphocytes (P b 0.05) without alteration in T lymphocytes Or CD4+/CD8+ ratio and number of CD3-/CD16+, CD56+ cells but decreased NK activity(P b0.05). The study of other factors is neede to detect those who are more suseptible to infections.Immuno-Oncology. Objectives: Like p16, p15 is a gene which inhibits CDK4 and CDK6 and negatively regulates the cell cycle. Because DNA methylation of p15 is frequently observed in patients with acute myelogenous leukemia (AML), the silencing of p15 is thought to be closely related to the onset and progression of tumors. The present study was undertaken to examine the relationship between DNA methylation of p15 gene and histone modification in AML. Methods: Tumor cells were isolated from the bone marrow or peripheral blood of 6 patients with AML (4 cases of primary AML and 2 cases of recurrent AML; 4 boys and 2 girls with ages ranging from 1 to 14 years). More than 90% of the isolated cells were confirmed to be leukemic cells. Bone marrow and peripheral blood mononuclear cells from healthy individuals were used as control. Leukemic blood cell line, HL60, which expressed p15 gene, and MOLT4 whose p15 was silenced by methylation were also examined. Acetylation of histones H3 and H4 and the methylation of histone H3 at lysine 9 (H3 Lys 9) were analyzed by chromatin immunoprecipitation. The methylation status of p15 gene of immunoprecipitated DNA was evaluated by PCR using methylation specific primer. Results: Bone marrow and peripheral blood mononuclear cells and HL60 cells showed acetylated histone H3 and H4, but not methylation of H3 Lys 9. On the other hand, MOLT4 cells had not only methylation of histone H3 Lys 9 but also histone H3 and H4 acetylation. Interestingly the p15 promoter DNA immunoprecipitated with antibody specific for acetylated histone H3 or H4 was unmethylated in some alleles and methylated in other alleles. Coexistence of methylated alleles and unmethylated alleles were observed by methylation-specific PCR and sequencing in the case of methylated H3 Lys 9. DNMTs expression levels were not overexpressed in AML, compared with their levels in normal bone marrow and peripheral blood. Biology in recent years has focused on identifying specific genes termed as Proto-Oncogenes, ex-pression of which appears to underlie the process of carcinogenesis. These Proto-Oncogenes become activated as Oncogenes by a variety of mechanism: viral transduction, promoter insertion, amplification, point mutations and chromosomal translocations. Out of which ras-gene belongs to Gprotein related to 21 KD, located on the inner face of plasma membrane (P21) is playing a very important role in detecting a wide variety of normal and tumour tissues. Immunological and Comparitive Study of Ras The present work was aimed at study of the distribution of rasoncogene in human epithelial cancers and precancerous lesions, with confirmed histological examination. Lesions were of oral cavity, respiratory tract, gastrointestinal tract and liver, breast, kidney and urinary bladder, ovary and skin. Cryostat sections of tissues were prepared. Primary antibody Ras (Ki/Ha) P21 (lyophilized) was obtained commercially and Peroxidase-Anti-Peroxidase method was used to localize the antigens.The staining was predominantly in the cytoplasm; appearance was fined granular/ diffused. Squamous cell carcinomas of various sites showed 95.6% positivity and 94.4% in cases of adenocarcinoma. Anaplastic carcinomas showed 71% positivity whereas cases of verruca, transitional cell, PagetTs disease showed 60% positivity. All the cases of benign lesions showed 65% positivity while normal tissues of oral cavity, respiratory tract and skin showed only 25% positivity.This study helps to compair the normal and abnormal tissues and also provides an excellent class of tumor markers for targeting and therapy, using immunological, pharmacological or radiolabelled agents to inhibit their functioning in cancers.Sa2.92. Defects in TCR Associated Proteins in Relation to Immune Impairment in Oral Cancer Patients.V. T. Cheriyan, 1 S. C. Dutt, 2 S. M. Krishna, 1 P. Balaram. 1 1 Division of Cancer Research, Regional Cancer Centre, Trivandrum, Kerala, India; 2 Division of Radiotherapy, Regional Cacner Centre, Trivandrum, Kerala, India.Cancer of the oral cavity is easily detectable and curable in the early stages. However, recurrence and nodal metastasis are frequent often due to late diagnosis of the disease and limited understanding of the biology of the tumour. Immunosuppression is reported in cancer patients even though the mechanism remains illdefined. Recent reports suggest T-lymphocytes to be the principal effector cells in this process and modification of signal transducing molecules to be responsible for the impairment. T-cell activation also leads to translocation of NFkB components like Rel-B and c-Rel to the nucleus activating the transcription of variety of genes including IL2. This study addresses the expression status of the signal transducing proteins and its role in the immune impairment in patients with cancers of the oral cavity. 70 oral cancer patients were collected for the study, after obtaining informed consent. Healthy normal individuals were selected as controls. Percentage population of CD3 +, CD4+ and CD8+ cells were enumerated by surface phenotyping using FACS Calibur and capacity to respond to mitogens and anti-CD3 was assessed by the Thymidine incorporation assay. T-cell function was assessed by quantitation of levels of IL-2 production after stimulation with PHA/anti-CD3. The expression status of the signalling molecules TCR-~, CD3-e, zap-70, p 56 lck, PKC, rel-B and c-rel was evaluated by western blotting following stimulation of lymphocytes with anti-CD3 in presence and absence of r-IL2. Surface phenotyping of CD3 +, CD4+ and CD8+ cells showed significant decrease in CD3 and CD4 positive cells and the CD4/CD8 ratio with significant increase in the CD8+ cells. Cell proliferation assay clearly demonstrated defects in cell proliferation to T cell specific mitogen and majority of the oral cancer patients showed decreased response to PHA and anti-CD3. Production of IL-2 by the lymphocytes was also found to be reduced. Exogenous Interleukin-2 could increase the transformation response in all the controls and in 20% of the patients to different degrees varying from 10% to 90%. Low levels of the signaling molecules (TCR-~, CD3-e, zap-70, p 56 lck, PKC, rel-B and c-rel) and an impairment in the transduction of rel-B to the nucleus were observed in these lymphocytes. Decreased CD4 + / CD8 + ratio with an increase in suppressor cells, reduced lymphocyte proliferation and production of IL-2 suggest a defective immune regulation in cancers of the oral cavity. Impairment in the translocation of rel-B to the nucleus and the reduced levels of signal transducing proteins might be the reason for the decreased production of interleukin-2 and immune impairment in oral cancer patients. Is the Oncogenesis Epigenetic Induced Alternative Way for Cell Survival? 1 Ob/Gyn, Medica Centre, Nis, Serbia, Yugoslavia; 2 Department for Byology, Medical University School, Nis, Serbia, Yugoslavia.Microevolution of tumors is accompanied by all, or almost all, elements that characterize evolution in the Darwinian sense. The evolution of tumors progress on a time scale of months and years within a limited population of altered cells, involve all the phenomena observed in the long-term evolution of species. These phenomena include random changes in the genome of tumor cells, various forms of selection pressure and selection of tumor cells. Mutations of key genes endow tumor cells with a selective growth advantage and, in the presence of an unstable genome, these cells further mutate and undergo progressive dclonal evolutionT. Albeit this model of tumor survival is widely accepted, there is possibility that emergence of dfirstT altered cell generation follow same axioms as mentioned phenomena of tumor growth and escape. To that effect, shifting of a normal cell to the tumor cells might bi explained as activation of adaptive mechanisms for cell survival. Thanks to large genetic potential and searching for dbetter solutionT, the dendangered cellT or cell under abnormal dconditions/selection pressureT may wend to malignant alteration as an alternative way for survival. For example, epigenetic factors such as dchemical distressesT or virus-infection may activate the silenced genes that are responsible for malignant alteration. In vertebrates, evolutionary accumulation of genetic potentials is associated with phenomena of epigenetic induced adaptation and ability of species for survival in different conditions. In the same way, shifting of the cell from normal to altered might be conceived as an epigenetic induced adaptation for cell survival, as well as mechanism of tumor escape.Sa2.94. Anti-Interleukin-10 Strategies in Treatment of Malignant Diseases.I. 1 Ob/Gyn, Medica Centre, Nis, Serbia, Yugoslavia; 2 Institute for Biology, Medical University School, Nis, Serbia, Yugoslavia.Interleukin-10 (IL-10) is important cytokine for phenomena of tumor growth, escape and even carcinogenesis. This cytokine is essential for tumor cell proliferation because its neutralization decreases clonogenicity of malignant cells, whereas addition of recombinant IL-10 increases cell proliferation. IL-10 autocrine/ paracrine loop plays an important role in the resistance of certain tumors to chemotherapeutic drugs. In addition, immunomodulatory/suppressive nature of IL-10 plays significant role in mechanisms of tumor escape from immune monitoring. Nevertheless, there is no clear anti-IL-10 strategy in treatment of malignant disease. In fact, there are a few hypotheses, and small number of experimental/clinical studies, that propose anti-IL-10 strategy in treatment of malignant diseases. Therefore, AS101 treatment combined with chemotherapy may be effective in the treatment of certain malignancies. Another possibility is treatment of malignant patients by anti-IL-10 antibodies with unpredictable consequences and complications in relation to autoimmunity and immune complexes disease. The strategy that we suggest is based on extracorporeal blood filtration/purification method with a view to removing IL-10 molecules from patientTs blood by anti-IL-10 antibodies attached on the filter walls. The present strategy provides a method for enhancing an immune response in tumor sufferers to facilitate the elimination of IL-10 and/or other immunosuppressive cytokines and other molecules. The method involves the removal of immune system inhibitors from the circulation of the tumor sufferers, thus, enabling a more vigorous immune response to the tumor. Particularly useful in the strategy is an absorbent matrix composed of an inert, biocompatible substrate joined covalently to a binding partner, such as antibody, capable of specifically binding to the IL-10. TNF-alpa is a pleiotropic cytokine which can induce apoptosis is sensitive cells, but also regulated cell proliferation, cellular activation and differentiation. To be better estimated role of TNF on PC cell line, originally developed from patients with myelodisplastic syndrome at Institute of Oncology Sremska Kamenice, Novi Sad, we monitored the kinetics of changes after in vitro treatment with or without TNF-alpha in presence anti-CD45 and CD95 MoAb, IL-3, FLT3 and GM-CSF. We analyzed cell viability by cell enumeration; intracellular metabolic activity by determination of total LDH activity after cell sonication, cell proliferation, cell membrane molecule expression, apoptosis and necrosis using flow cytometry (Becton Dickinson, San Jose, USA). Analyses were performed 2, 6, 8 and 24h after treatment under some experimental conditions. Our results showed that in comparison with untreated cells, TNF-alpha induced significantly increase in apoptosis and necrosis, in PC cells, which expressed high level of CD95 and TNF alpha receptors. Immunophenotype of this cells also excluded B cell or T cell charactristics, but presence of early hematologycal markers was found. Pretreatment of PC cell with anti-CD45 and anti CD95 monoclonal antibodies modulated cell death induced by TNF. In addition, presence of TNF in cell culture medium induced significantly decrease in cell proliferation, stimulated by IL-3, GM-CSF or FLT-3. However, no changes in CD13 and CD33 antigen expression following cell proliferation, determined after 7 days cultures and stimulation in comparison to percentage of antigen expression before treatment. No changes in intracellular LDH activity before and after cell proliferation induced with TNf or different cytokine kombinations. We conclude that sensitivity to apoptosis and evidence for their increase limited cell proliferation estimated on this cell line. Introduction: The ability of T cells to recognize a tumor antigen is the most important step in immune activation. The degree to which this event occurs in the CNS remains a matter of open debate. In the present study we sought to (1) determine whether peripheral T cell priming occurred in response to intracranial tumors, (2) to evaluate whether the degree of priming offered a survival advantage, and (3) to design ways of improving the specificity and delivery of T cells to intracranial tumors.Methods: The B16/F10 metastatic melanoma cell line was transduced with the H-2K b restricted SIY peptide. 1Â10 3 or 10 6 B16-SIY cells were the implanted in the flanks or brains of H-2K b restricted C57BL/6 and 129S6 mice. ELISPOT assays were performed on spleens isolated 7 or 13 after tumor implantation to assess endogenous T lymphocyte responses. In a separate set of experiments, naRve 2C CD8 + cells, which recognize the SIY peptide, were isolated from 2C/RAG2-/-mice by negative selection and stimulated on Days 0 and 4 through co-incubation with replication-incompetent, H-2L d -and B7-co-expressing P815 mastocytoma cells in a 1:5 ratio. The cells were then injected into B6/129 mice harboring B16-SIY intracranial tumors with or without local intracranial delivery of IL-2 at varying time points.Results: While the B16-SIY tumor grew in the flanks of C57BL/6 mice and was associated with poor T cell priming to the SIY antigen, it did not grow in 129 mice where the T cell response was prominent. In contrast, intracranial delivery of B16-SIY was uniformly fatal in both animal models, in spite of peripheral T cell priming to the SIY antigen. Mice treated with tumor-specific T cells either before or after intracranial tumor implantation exhibited a statistically significant increase in survival, from 16 days (controls) to 21 days (T cells), P b 0.01. Co-therapy with T cells and IL-2 exhibited strong synergism: 16 days for controls, 39 days with T cell delivery prior to tumor establishment (P b 0.001), and 58 days with T cells after tumor establishment (P b 0.00001). Twentypercent of mice treated with T cells and IL-2 were long term survivors of greater than 120 days. Immunohistochemical analysis of animal brains revealed local CD4 + and CD8 + infiltration in intracranial tumor-bearing mice that had been treated with either T cells alone or T cells and IL-2.Conclusions: Our results confirm that T cell priming does indeed occur in response to CNS tumors, however, the strength of the response does not correlate with survival. While the use of tumor specific T cells offers a survival advantage, local delivery of IL-2 to the site of the tumor further augments the immunotherapeutic response. This study represents the first report in which T cells directed against intracranial antigens along with IL-2 exhibit a synergy which significantly prolongs the survival of animals with intracranial tumors. Most well characterized anti-angiogenic molecules are currently under clinical trials for cancer treatment. Most vertebrate CRTs are known by their chaperone properties. Since TcCRT is highly homologous to a functional antiangiogenic fragment from HuCRT (aa 120-180), recombinant (r) and native (n) TcCRT were tested in their antiangiogenic effects, in the chick embryonic chorioallantoid membrane (CAM) assay. This effect was further substantiated in experiments showing that the genetic plasmid construct pSecTag/TcCRT also displayed significant antiangiogenic properties, as compared to the empty vector, thus indicating that the parasite gene was transfected to the vertebrate host. Most likely, the fact that antiangiogenic substances act preferentially on growing neoplasic tissues but not on already established tumors, is due to their effects on emerging blood vessels. Thus, by virtue of its capacity to specifically bind to laminin, CRT may interfere with the assembly of endothelial cells and, as a consequence, in the generation of new blood vessels. In synthesis, our results indicate that TcCRT, like its human counterpart, has antiangiogenic properties. These properties may explain, at least partly, the reported antineoplasic effects of experimental MHC class I chain-related molecules (MIC) are NKG2D ligands that participate in immune surveillance of cancer. Engagement of NKG2D by cell surface MIC triggers NK cell activation and antigen-specific CTL immunity. However, the release of soluble forms of MIC (sMIC) by the tumor may impair this response by decreasing NKG2D surface expression. At diagnosis, around one third of patients showed increased serum levels of sMICA that correlated with white blood cell count and that decreased upon therapy with imatinib mesylate (IM), a specific BCR/ABL inhibitor. In K562 cell line, IM decreased both surface MICA/B expression and NKG2D-mediated lysis by NK cells. IM did not affect MICA mRNA levels but decreased MICA protein expression and sMICA release. Likewise, MICA expression was reduced upon treatment with inhibitors of PI3K and mTOR, that are both activated by BCR/ABL. Objective: To study the effect of MTX-loaded process on innate immune adherence activity to tumor cells of adult and cord blood erythrocytes.Methods: MTX was loaded in erythrocytes using the modified hypotonic preswelling technique. We used the erythrocytes after undergoing washing, swelling and drug-loading treatment from adult and cord blood erythrocytes, and measured the rate of rosette formation.Results: We observed that the innate immune adherence activity of erythrocytes from adult and cord blood after hypotonic swelling treatment significantly increased as compared with other groups ( P b 0.05), which the rate of rosette formation was up to (61.45 F 8.51)%, (17.00 F 2.16)%, respectively. However, MTXloaded treatment was no significant change than un-treated carrier erythrocytes.Conclusion: MTX-loaded treatment did not affect the innate immune adherence activity to tumor cells of erythrocytes significantly. Objective: To research the potential role of erythrocytes in leucocytes reactivity against with neoplasms.Methods: 0.2ml suspension of inactivated cancer cells (S180, 5Í10 6 /ml) (or 0.2ml NS as control) were added into 0.2ml fresh anticoagulant whole blood (or 0.2ml white blood cells) treated by citric acid, and incubated for 1 h at 378. Using monoclonal antibody of CXCR4, we compare the difference of expression level of CXCR4 in leucocytes, and compute the activation index (treated group-control group/control group).Results: It was found that cancer cells could activate hematogenic immunity. In 8 normal blood donors and cancer patients, the activation index of CXCR4 in whole blood cells was significantly higher than that in white blood cells (t = 2.598, P b 0.05).And in the cancer groups, the modulus of CXCR4 (57.64 F 10.30) in whole blood cells treated group was significantly higher than that (35.26 F 4.92) in whole blood cells control group (t = 3.397, P b 0.01).Conclusion: These results indicate that there is a road map of blood immune reaction and erythrocytes is requisite. The erythrocytes can promote the expression of CXCR4 of granulocyte. Erythrocytes plays a vital role in leucocytes reactivity against with neoplasms. Objective: To establish a detection method of IL-8, IL-6 and DARC (Fy6), Which has application in the study of cancer cell activized to hemeimmune reaction line map theory. Cancer Cell Activation Study of Hemaimmune Methods: Using ELISA method of IL-8. We detected Experimental system of hemaimmune reaction road map. Cancer cells (S180,5Â10 6 /ml) or NS were added in fresh anticoagniant whole blood (treated by citric acid), or in red blood cell and white blood cells and plasma (or NS),and incubated for in at 378 to see results.Results: Cancer cells (deathly cells) can activate hemaimmune reaction road map experimental system. In cancer cells added to whole blood group, level (531.6pg/ml) IL-8 higher than that (b0.5pg/ml) In NS added to whole blood group and in cancer cells added to whole blood group, Level (28.85) of Fy6 on red cell Lower than that (45.80) of Fy6 on NS added to whole blood group. In cancer cells added to whole blood cells group, Level (2.86 or 1) of activation index (IL-8 or IL-6) higher than that (0.36 or 0.25) in cancer cells added to white blood cells group. In cancer cells added to white blood cells group, Level (138-126.3pg/ml) of IL-8 higher than that (55.99-3.90pg/ml) in NS added to white blood cells group.Conclusion: The results suggested that the red blood cells and complement plays a vital role in regulation (IL-8, IL-6) of hema immune reaction. And there is road map of hemaimmune reaction. Antigen (cancer cells) can activate complement, Adhere to complement (C3b), than be adhereing to red blood cells, Then adhereing to white blood cells to activate hemaimmune reaction. Furthermore, it can provide useful experimental system of hemaimmune reaction road map theory study.Sa2.102. Apoptosis-Based Therapies for Hematological Malignancies. Apoptosis is an intrinsic cell death program that plays critical roles in tissue homeostasis, especially in organs where high rates of daily cell production are offset by rapid cell turnover. The hemopoietic system provides numerous examples attesting to the importance of cell death mechanisms for achieving homestatic control. Much has been learned about the mechanisms of apoptosis of lymphoid and hematopoietic cells since the seminal observation in 1980 that glucocorticoids induce DNA fragmentation and apoptosis of thymocyte and the demonstration in 1990 that depriving Colony Stimulating Factors (CSFs) from factor-dependent hematopoietic cells causes programmed cell death. From an understanding of the core components of the apoptosis machinery at the molecular and structural levels, many potential new therapies for leukemia and lymphoma are emerging. In my presentation, I will introduce some of the drug discovery targets thus far identified within the core apoptotic machinery, and describe some of the progress to date towards translating our growing knowledge about these targets into new therapies for cancer and leukemia. CD4+CD25+ T cells may play an important role in mediating immune tolerance by regulating T cells which cause autoimmune disease. Evidence from murine systems suggests hese cells inhibit immune responses against tumours. In order to investigate this hypothesis, we have decided to assess their role in leukemia relapse after either bone marrow transplantation and/or chemotherapeutic treatment. Leukemias and patients were selected for this project. Immunophenotypic analysis indicates that patients with CML who relapsed after allogenic BMT have a higher proportion of CD4+CD25+ T cells compared to those who are disease free. Our findings suggest that a proportion of CD4+CD25+ T cells in CML patients at relapse are inhibitory. Therefore, these results may reveal that a relapse after chemotherapeutic treatment may be caused by an increase in the regulatory population which may lead to the enhanced inhibition of a graft versus leukemia effect. Gluten intolerance is system immunological disorder which is characterized in part by the presence of antigliadin antibodies, which sometimes are also directed to calreticulin. The aim of this work was to determine is there any immunoreactivity of serum IgA with gladin, or with tissue transglutaminase, in small group of patients with myeloma multiplex.Three patients with IgA and 4 patients with IgG myeloma multiplex, 1 patient with both IgG and IgA, M components in the serum, 1 patient with non-Hodgkin lymphoma-lymphoplasmocyticum, and 7 healthy people were included in the study. For determination of the level of the immunoreactivity of antigliadin IgA antibodies a home made ELISA test was used, with 5 micrograms of crude gliadin (SIGMA) as the antigen, while 1% bovine serum albumin was used as blocker. Immunoreactivity of IgA anti-gliadin antibodies was determined in prediluted serum to 1:250, and 1:500. Blanks were wells coated with blocker 1%BSA (without gliadin), incubated with corresponding serum dilutions, treated with secondary, HRP labeled, goat anti-human IgA antibody, and additionally, wells coated with only gliadin treated with secondary, goat anti-human IgA antibody. Immunoreactivity of IgA anti-transglutaminase antibodies was determined in prediluted human serum to 1:100, using commercial. ELISA test (Binding Site), with recombinant tissue transglutaminase, as the antigen.Results from this work showed high intensity of the interaction of serum IgA with gliadin in two patients with IgA plasmocytoma (only in patients with IgA, M component in the serum). The OD492 at 1:250 serum dilution were 0.523 and 0.206, while there was no interaction of serum IgA with gliadin in all healthy persons sera, i.e., at 1:250 serum dilution it was in the limits of the experimental error (OD492 b 0.050). The level of this reaction was of less pronounced intensity, in one patient with IgA plasmocytoma (in his serum there was no M component), OD492 was 0.107. In one patient with non-Hodgkin lymphoma-lymphoplasmocyticum, antigliadin IgA immuno-reactivity was high, OD492 was 0.750. Surprisingly, patient with two M components, showed IgA immune reactivity with the blocker, bovine albumin, but not with gliadin.There was no intensive reaction of serum IgA with tissue transglutaminase for all examined patients, indicating that all immune disturbances characteristic for celiac disease are not developed in these patients.In conclusion, these preliminary results are the first showing antigliadin IgA immunoreactivity in some patients with myeloma multiplex; they set up a question on the importance of gluten intolerance, (or of some parasitic/or bacterial infection which could induce anticalreticulin/antigliadin immunity ?) in the emergence and development of myeloma multiplex. Ikaros gene family encodes for a group of Kruppel-like zincfinger proteins which act as lymphoid-specific transcription factors. Knock-out studies on mice have shown that Ikaros, Aiolos and Helios function as transcription regulators playing an important role in differentiation of particular subsets of lymphocytes.All members of Ikaros gene family share the similarity in their overall structure. The four N-terminal zinc-finger motifs are responsible for sequence-specific DNA-binding, while C-terminal zinc-finger pair acts as a dimerization domain: homo-and heterodimers of Ikaros, Helios and Aiolos bind DNA with high affinity in a sequence-specific manner. These complexes can then activate or repress transcription from certain promoters if the DNA-binding domains of both members of the complex are intact. However, by the mechanism of alternative splicing different isoforms can be created that lack one or more Nterminal zinc-fingers. There is a possibility that up regulated production of shorter isoforms could result in phenotypes seen in knock-out mice which include complete or partial arrests in different stages of lymphocyte development, leading to development of leukemia and lymphomas. Therefore we decided to correlate the expression of long and short isoforms of these proteins in bone marrow, peripheral blood lymphocytes and lymph nodes of patients with different lymphoproliferative disorders. Using reverse-transcription-polymerase chain reaction (RT-PCR) we analyzed lymph nodes from patients with different types of leukemia. Eight human hematological cell lines were also screened for the expression of Aiolos and Helios. Preliminary results show a heterogenous pattern of expression of Aiolos and Helios among these samples. Shorter isoforms, which are probably generated in smaller amounts than fully functional molecules, will be detected by more sensitive real-time PCR and different subpopulations of cells will be analyzed using flow cytometry. The results could improve our understanding of the mechanisms of normal lymphocyte differentiation and possible pathways in development of human lymphoproliferative disorders. The heterogeneity of effector functions displayed by Rituximab and other anti-CD20 mAb which apparently recognize the same CD20-epitope suggests that additional mechanisms, probably related to mAb fine specificity, can be responsible for B cell depletion. To improve our understanding of Rituximab function, we investigated its fine specificity. The biopanning with Rituximab of an eptapeptide cystein-constrained (c7c) phage display peptide library (c7c PDPL), of an eptapeptide linear (7-mer) PDPL, and of a dodecapeptide linear (12-mer) PDPL resulted in the isolation of 13, 9 and 10 clones respectively, which specifically reacted with Rituximab in ELISA and Western blot. The c7c PDPL-derived clone insert sequences expressed the motif A(S)NPS, which matched the CD20 amino acid stretch 170 ANPS 173 . Two cyclic peptides bearing ANPS-(Rp15-C) and SNPS-(Rp3-C) motif specifically recognized Rituximab paratope, while no reactivity was observed with Rp3-C-derived peptide mRp3-C (bearing the mouse CD20 170 SNSS 173 ). The 7-and 12-mer PDPL-derived clone insert sequences expressed the motif WPXWLE, which could be aligned only to the reverse-oriented amino acids 161 WPKWLE 156 of acid sphingomyelinase-like phosphodiesterase 3b precursor (ASMLPD3b), though linear peptide Rp1-L, Rp5-L and Rp10-L bearing the motif competed with cyclic peptide for Rituximabparatope binding. Furthermore, Rituximab reacted with ASMLPD3b-derived peptide, suggesting a possible interaction with this enzyme precursor. Our results indicate a unique fine specificity of Rituximab and suggest a possible mechanism to explain the recently reported ability of Rituximab to increase acid sphingomyelinase activity in raft microdomain. Functional Analysis of Peripheral Blood Lymphocytes Subsets in Patients with Cancer-Associated Dermatomyositis. A. Ponyi, 1,2 M. Aleksza, 1 L. Gergely, 1 M. Garami, 2 T. Constantin, 2 K. Danko. 1 Objectives Dermatomyositis is characterized by immune-mediated muscle inflammation leading to progressive weakness of the skeletal muscles and the presence of cutaneous symptoms. Patients with dermatomyositis have a higher risk of malignant disease than the normal population. Patients and Methods Our aim was to characterize different lymphocyte subsets in cancer-associated dermatomyositis patients compared with healthy controls and patients who had primary dermatomyositis. Fifteen patients with cancer-associated dermatomyositis and 44 primary dermatomyositis patients were included in this study. Different lymphocyte subpopulations were measured by phenotypical characterization with monoclonal antibodies. Intracellular cytokine expression of peripheral T lymphocytes was assessed after an in vitro incubation with an activating cocktail. The cells were labeled with anti-CD4 or anti-CD8 antibodies and intracellular accumulation of IL-4 or IFN-g was detected. The frequency of different Tcell subsets was measured within the T lymphocyte population by flow cytometry. Results Concerning on the phenotypic characterization, there were no detectable differences in the percentages of CD4+, CD8+ and CD19+ cells. Compared to controls, CD3+ cells were decreased (70.9% vs. 56.6%), while CD56+ cells were elevated (8.4% vs. 15 .1%) in cancer-associated dermatomyositis patients, but not in primary dermatomyositis patients. The ratio of activated CD3+/ HLA-DR+ and CD3+/CD69+ cells was elevated both in cancerassociated and primary dermatomyositis cases. There was no significant difference in the ratio of Th1 cells compared to controls both in untreated and treated cancer-associated dermatomyositis patients (21.0% vs. 24.4% and 21.9%). On the other hand, significantly decreased Th1 ratio was found in active primary dermatomyositis patients (21.0% vs. 9.8%). The percentage of Th2 cells were normal in cancer-associated and in primary dermatomyositis patients as well, but it was decreased in inactive primary dermatomyositis patients compared to healthy controls.Conclusion T lymphocytes of primary dermatomyositis were less polarized towards Th1 cells, while this was not observed in cancer-associated dermatomyositis patients. Cancer-associated dermatomyositis differs from primary myositis in many aspects of clinical and immunological features. Better understanding of the precise interaction of the host immune system with the malignant disease can shed light on the association between an autoimmune disease and malignancy.Sa2.108. Bacteriophage HAP1: Molecular Basis of Its Antitumour Activity. Previously we investigated interactions of bacteriophage T4 with mammalian cells: unexpected binding of phage T4 to cancer cells and its antitumour activity. We selected in vitro the mutant HAP1 that was able to bind the cells much stronger (than parental T4), it was also more effective against B16 melanoma tumour and metastases in vivo (Dabrowska et al. Anticancer Res. We proposed a possible molecular basis of these interactions: reactions of beta3 integrins on target cells and T4 capsid proteins: probably gp24, which contain KGD-aminoacid motifs, i.e. RGD homologues able to bind beta3 receptors (Gorski et al., Medical Immunol. Here we present further results that may highlight non-antibacterial properties of phages.In direct sequencing of phage-head genes we found a non-sense mutation in the hoc gene of HAP1. Phage particle size and morphology was observed in the electron micrographs and determined by dynamic light scattering (PCS). T4 and HAP1 do not differ in their general morphology, but the head of HAP1 is smaller than the head of T4. This is in line with the well-described morphogenesis of the T4 capsid: after incorporation of Hoc protein T4 phage head becomes visibly larger. The normal Hoc protein is balloon-shaped and it extends to about 5 nm away from the capsid surface, 160 regularly arranged units per one capsid. Because of its special localisation gp Hoc impedes access of external factors to the head surface. Without gp Hoc, there are no important spatial disturbers that can diminish the interactions of other head components with any external targets. This also applies to gp 24, which was proposed as the active protein.Differences in T4 and HAP1 interactions with platelets were also observed (Kniotek et al., Immunology 2004) . However, a very interesting report reveals its relatedness to eukaryotic immunoglobulin-like domains. This similarity results rather from divergence from a common ancestor, not just from convergent evolution (Bateman et al., Virus Genes. The immunoglobulin superfamily is engaged in adhesion processes, MHC functions, Tcell receptors, and others. On the other hand, we know that higher organisms are strongly exposed to bacteriophages. They have become ban environmentQ for phage life cycles (Dabrowska et al., J. Appl. 98, 2005) and, one can expect, phages adapt to them. All these considerations suggest that some bacteriophage molecules are predicted to interact with eukaryotic organisms, influencing important immunological processing and/or to modulate these interactions. Objective: Although immune response is thought to regulate the progression of various cancers, cancers often progress by escaping from hostsT immune surveillance. In the present study, we investigated the changes in the immune status during the progression of mouse leukemia.Materials and Methods: We established a leukemia model by injecting in BALB/c mice with WEHI-3b cells intraperitoneally. Mononuclear cells were isolated from peripheral blood (PB) and bone marrow (BM) every 10 days, and analyzed the cell composition by flow cytometry. Cells were separated by using magnetic microbeads-conjugated antibodies.Results and discussion: The numbers of peripheral white blood cells (most of them were leukemic cells) were dramatically increased after day 24 following injection of WEHI-3b cells, and reached 1.8Â10 5 /ml on day 30. During progression of leukemia, both dendritic cells (DCs, I-A d CD11c + ) and DX5 + CD3-cells showed marked increases in PB, although most cell types were increased. We, therefore, focused on the kinetics and functions of those two cell populations. Increased DCs expressed lower levels of I-A d than that of DCs from normal mice and had low allo T-cell stimulatory activity. In the BM of leukemic mice, only DX5 + CD3-cells were continuously increased despite the progression of leukemia, and those cells were rapidly increased in PB. The increase in DX5 + cells in BM was thought to be induced by soluble factors from leukemic cells, because co-culture of WEHI-3b cells with normal bone marrow cells in trans-wells showed a selective increase in DX5 + and CD25 + cells. The morphology of most of the circulating DX5 + cells from leukemic mice was large granular lymphocytes, and the surface markers of the cells were shown to be lineage-CD94-CD122 + CD25 + Ly-49-Thy-1 bright c-kit dim . These data suggest that those DX5 + cells were immature NK cells. Isolated circulating NK cells from leukemic mice were able to down-regulate the expression of I-A d on normal BM-derived DCs, which was possibly mediated by TGF-h produced by those cells. Moreover, these NK cells significantly suppressed the allo T-cell stimulatory activity of DCs, which required cell-to-cell contact between NK cells and DCs, and CD25 was thought to be involved. Because direct interaction between NK cells and DCs induced IL-2 production, IL-2 might be associated with the inhibitory effect. In the d90s, Thy-1 + bone marrow cells were reported to have immunoregulatory functions in bone marrow transplantation without further analyses. Immature Thy-1 + NK cells with immunosuppressive activities identified in the present study might be a population of such cells.Conclusion: We have identified circulating immature NK cells with immunosuppressive activities during the development of leukemia, which is important not only for understanding the development of the disease, but also for effective immunotherapy. The Application of an In Vitro Viability Assay Followed by Subtractive Hybridization for Identification of Prognostic Markers in Acute Myeloid Leukemia. 2 A high percentage of acute leukaemia patients relapse after an initial response to treatment. Currently, risk-adapted therapy is useful for only a fraction of patients. Novel markers need to be identified to provide therapeutic direction to reduce over-treatment or under-treatment of patients. Furthermore, the mechanisms that regulate early relapse have not been identified. A significant correlation (r = À.721, P = 0.005) between viability and duration of survival was observed. We then selected a sample with low in vitro viability and a longer survival period and another sample with high viability and short survival period. Subtractive hybridization was performed to enrich and amplify for differentially expressed genes in these two samples. Nucleotide sequencing of four preliminary clones showed one differentially expressed gene was a mitochondrial gene, cytochrome c oxidase. This gene was upregulated in a sample that showed high in vitro viability and shorter survival period. Mitochondrial genes have been indicated in leukaemia patients refractory to conventional chemotherapy and may play a role in early relapse. Thus, we conclude that in vitro cell viablity may be useful as a prognostic marker and the study of this biological difference in acute leukemia patients may lead to a further understanding of the mechanisms that control cell survival and relapse. Background: Cervical carcinoma is a human papilloma virus (HPV)-related malignancy, in which escape of the tumor from the hostsT immune response is thought to play an important role in carcinogenesis and may involve alterations in the expression of immune-regulatory molecule genes. The purpose of the present study was to evaluate the relationship between the expression levels of CD28, cytotoxic T-lymphocyte-associated-4 (CTLA4), inducible costimulator (ICOS), ICOSL, CD80 (B7-1), CD86 (B7-2) and Granzyme B (GrB) genes and response to treatment in cervical cancer. Materials & Methods: The mRNA levels were determined, by quantitative real-time RT-PCR in cervical cancer specimens from 75 patients collected prior to radiotherapy or radiotherapy plus chemotherapy. After 6 months, the patients were divided into two groups, according to disease persistence (n = 25) or not persistence (n = 50). Target mRNA levels were normalized by the mRNA level of reference gene (DHPS) and expressed in relative units (RU). Results: GrB and CD86 mRNA levels were significantly higher in biopsies from patients with persistence than in those without persistence, with median values of 2.12 and 0.84 RU for GrB (P = 0.008), 0.53 and 0.27 RU for CD86 (P = 0.001), respectively. No difference was observed regarding the other molecules. Conclusion: Besides prognostic potential concerning response to treatment, this finding also suggests association of more aggressive tumor process with a particular immune activation profile. Intratumor BCG administration can eradicate local as well as metastasized cancerous cells, suggesting development of immunity against the tumors. Administration of BCG into noncancerous normal tissues, however, results in no significant effect on the tumors. A potent inflammatory agent, BCG, in normal tissues activates membranous phospholipase A 2 to release lysophospholipids that efficiently activate macrophages. Because cancerous tissues contain alkylphospholipids, BCG-induced inflammation produces alkyl-lysophospholipids and alkylglycerols that activate macrophages with approximately 400 times more efficiently than lysophospholipids (Yamamoto et al. Cancer Res 48: 6044-9) . These results imply that highly activated macrophages can kill cancerous cells. Inflammation-primed macrophage activation process is the principal macrophage activation cascade that requires serum vitamin D 3 -binding protein (known as Gc protein) and participation of B and T lymphocytes. Stepwise hydrolysis of Gc protein with h-galactosidase of inflammation-primed B cells and sialidase of T cells yields a potent macrophage activating factor (MAF), the protein with N-acetylgalactosamine as the remaining sugar. Stepwise treatment of highly purified Gc protein with immobilized h-galactosidase and sialidase generated the most potent MAF (terminated GcMAF) ever discovered which produces no side effect in human. Administrating 10 pg GcMAF per mouse and 100 ng GcMAF per human results in the maximal activation of macrophages, which develop enormous variation of receptors. When human macrophages were treated in vitro with 100 pg GcMAF/ml for 3 hrs, the macrophages were highly activated. These macrophages can bind a variety of cancerous cells. Because cancer cells are too big to be phagocytized, macrophage membrane spreads over the cancer cell. These attached macrophage membrane surface is the same as phagosome membrane, which secretes superoxide and other lytic enzymes. Trypan blue begins to penetrate through the attached site and eventually stains the entire cells. Time course study of the cell death was performed with effector/target ratio of 3. In 4 hrs, 51% of prostate cancer cells LNCaP and 60% of breast cancer cells MDA-MB-231 were killed. In 18 hrs, 82% of LNCaP and 86% of MDA were killed. When prostate, breast, colon and lung cancer patients were treated with less than 25 weekly administrations of 100 ng GcMAF, the majority of cancer patients, excluding very advanced, exhibited healthy control levels of the serum prognostic index, indicating eradication of tumors. When in vitro cancer cell-killing study with macrophages activated by GcMAF was performed in the presence of serum or IgG fraction of GcMAF-treated prostate and breast cancer patients, greatly accelerated cell-killings (more than 80% of cells in 4 hrs) were observed. These results suggest that the macrophages activated with GcMAF kill cancerous cells via Fc-receptor mediation preferentially and that GcMAF-therapy develops IgG antibodies against the tumors. Light chain amyloidosis (AL) is a plasma cell neoplasia, where there is a clonal expansion of terminally differentiated B cells (plasma cells) within the bone marrow (BM) and the monoclonal immunoglobulin light chain forms insoluble amyloid fibrils that deposit systemically. We have previously shown that there is a bias in light chain variable (VL) gene usage in clonal plasma cells, with 3 rarely used E light chain genes (6a, 3r and 2b2) accounting for 60% of the AL light chain V gene repertoire. We now show that peripheral blood and bone marrow of these patients contain clonally related B cells, that have a mature B cell, non-plasma cell phenotype, in the absence of plasma cells. We evaluated the light chain V gene repertoire in the clonal isotype compartment (n or E) of 5 AL patients (3E + 2n for PBL and 4E + 1n for BM) and 2 healthy controls. The BM mononuclear cells were enriched separately for CD19+ B cells and CD138+ plasma cells. The peripheral clonal isotype repertoire had between one-third to onehalf of the B cells using the same V and J genes as the clonal population in the BM. By sequence alignment of the blood B cells and BM plasma cells using the same VJ genes, there was evidence of intraclonal variations in the blood B cells compared to the BM cells. Also, there was a restriction in diversity in the VL gene repertoire with AL patients using 6-8 different genes compared to 12-16 VL genes in the healthy controls. Though there is a clonal expansion within the specific isotype compartment, there is no change in the global numbers of n and E B cells in blood. In the CD19+ BM population, 3 of the 5 patients tested had between 0.8-1.4% of plasma cells in the B cell population. Three of the 5 patients had between one-third to greater than two-thirds of the CD19+ B cells in the BM using the same VJ genes as the clonal CD138+ (plasma cell) population. In the remaining 2 patients, one had no evidence of any clonally related CD19+ B cells while the other showed 3 populations of B cells using the same V gene but different J genes. In this latter patient, we were unable to identify a dominant clonal plasma cell population. Interestingly, the greater the number of clonal B cells in the CD19+ BM population, the more restricted the VL gene diversity. The CD138 and CD19 sorted populations in the BM from the healthy controls showed a typical polyclonal repertoire with 22-25 VE and 15-17 Vn genes represented in the CD138+ plasma cells and 7-15 VE and 15-23 Vn genes in the CD19+ B cells. The presence of clonal B cells in the BM and blood of AL patients is relevant for understanding the pathobiology of disease and also has significant implications for therapy. Immunomodulation by Different Forms of a Chimeric Costimulatory Molecule That Selectively Binds to CD28. The efficacy of several immunotherapeutics and adjuvants is limited by their specific activity and/or undesired ligand binding properties. We have used DNA shuffling to generate large libraries of chimeric adjuvant proteins and costimulatory molecules, and have selected the improved variants based on ligand binding and functional properties. This presentation describes one such chimera: CD28 binding protein (CD28BP), which is a DNA shuffled version of B7.1 that preferentially binds to CD28. Interestingly, CD28BP demonstrates adjuvanticity in a membrane-bound form yet inhibitory activity when soluble. We have combined DNA encoding CD28BP with DNA encoding an in silico shuffled cancer antigen that induces immune responses cross-reactive with Epithelial Cell Adhesion Molecule (EpCAM) to form the basis of a cancer vaccine candidate. In non-human primate studies we show that this approach achieves the goal of augmenting EpCAM-specific immunity with an excellent safety profile. Importantly, we demonstrate that the presence of CD28BP in the vaccine construct is required for the detection of EpCAMspecific CD8 T cell IFN-gamma production. Based on these findings, we believe this novel vaccine approach has the potential to break the immunological tolerance against EpCAM that limits current colorectal carcinoma vaccine approaches. Moreover, CD28BP has broader applications as a general DNA vaccine adjuvant. In contrast, soluble CD28BP protein has the capacity to inhibit mixed lymphocyte reactions and antigen-specific T cell responses in vitro making it a suitable candidate for autoimmune therapeutic applications. For the activation of naRve T cells two signals are necessary. Besides the cognate recognition of antigen in the context of MHC molecules, co-stimulatory signals are essential for effective T cell responses and here the CD28/B7 receptor ligand system is best characterized. The role of CD28/B7 for anti-tumor immune responses is still controversial. We induced anti-Trp-2 180-188 /K b melanoma specific CD8+ T cell responses by DC vaccination and compared their efficacy to control either subcutaneous or pulmonary metastases of B16 melanoma in CD28-deficient and wildtype mice. In both models, the tumors developed faster in CD28deficient mice. For subcutaneous metastases, the difference was only prominent during a certain time window after tumor challenge, whereas the areal tumor burden of pulmonary metastases was significantly enhanced in CD28 k.o. Furthermore, the influence of CD28 signalling on priming, homing or effector function of Trp-2 180-188 /K b -reactive circulating or tumor infiltrating T cells was phenotypically investigated. Trp-2 180-188 /K b -reactive CD8 + T cells could be detected after DC vaccination in a comparable frequency in the circulation as well as among the cellular infiltrate in subcutaneous tumors in both geneotypes. Clonotype mapping of tumor infiltrating lymphocytes showed that subcutaneous tumors in animals of either genotype harbored an oligoclonal infiltrate. Functional analysis of Trp-2 180-188 /K b -reactive cells, however, revealed that the number of IFNg-producing cells was substantially lower in CD28 k.o. Hence, in our model priming or homing of Trp-2 180-188 /K b -reactive CD8 + T cells does not seem to be affected in CD28 k.o. mice, but CD28 seems to be essential for effector functions of tumor specific CD8 + T cells. ALL and CLL Cells Express Elevated TNF-alpha upon Stimulation When Compared with Normal B Cells. 1 1 Special Diagnostic Immunology Laboratory, Hackensack University Medical Center, Hackensack, NJ, USA.Rationale: We hypothesized that the malignant B cell has significant autocrine function inducing its own replication and sought to establish its role in its own survival by evaluation of its cytokine profile compared with B cells from phenotypically normal (NL) peripheral blood (PB) and bone marrow aspirates (BM). We further hypothesized that the immunologic profiles differ by the type of B cell malignancy.Methods: Discarded BM or PB specimens from patients diagnosed morphologically and immunophenotypically as ALL (n = 16) or CLL (n = 18) were evaluated for induced expression of tumor necrosis factor alpha (TNF), IL2 and IL4 and compared with NL BM or PB specimens (n = 13). Specimens were washed, resuspend at 1-10Â10E6/ml in RPMI+10% a FCS and incubated with PMA (25 ng/ml), ionomycin (1.0 mcg/ml) and brefeldin A (10mcg/ml) for 4-6 hours at 378 in 5% CO 2 . The cells were then washed, resuspended in PBS, incubated with monoclonal antibodies (MAb) to CD19; CD2; CD38 and CD45 (Beckman Coulter, Hialeah FL) for 15 minutes, then fixed and permeabilized with Intraprep kit (Beckman Coulter) and incubated with MAb to TNF, IL2 and IL4. The cells were washed, resuspended in PBS and analyzed by a Coulter XL flow cytometer using standard CD45 by right angle side scatter employing logical gating strategies using PE and FITC conjugated isotypic controls antibodies to establish the histogram region negative for fluorescence. The percentage of CD19+ cells expressing TNF was recorded for each specimen. The mean for each of the ALL and CLL groups was compared with the NL group using a standard t test with unequal variance.Results: Stimulated ALL cells showed a statistically significant increase in the percentage of cells expressing TNF (7.25%) compared with NL stimulated B cells (0.3%) (P = 0.012). Similarly, stimulated CLL cells showed a statistically significant increased expression of TNF (4.06%) compared with NL stimulated B cells (P = 0.015).The difference in TNF expression between stimulated ALL cells and CLL cells did not reach statistical significance (P = 0.17). Within the CLL group, for specimens with B cell CD38 expression less than 15% (n = 9) the mean TNF expression was 0.68% and for specimens with CD38 expression greater than 15% (n = 6) the mean TNF expression was 10.6%. There was a significant difference in the percentage of TNF expression in those CLL cells with CD38 expression greater than 15% compared with those with CD38 expression less than 15% (P = 0.04). There was no difference between NL cells and ALL or CLL cells and induced IL2 and IL4 expression.Conclusion: Stimulated ALL and CLL B cells express TNF suggesting a possible role for this cytokine in the pathogenesis of these malignancies. These findings support the initiation of an investigation into the possible role for available anti-TNF therapies in clinical studies of both ALL and CLL.This work is support by a grant from the National Leukemia Research Association. Efforts to enhance anti-tumor immunity have led to identification of numerous tumor-associated antigens, but vaccination with such antigens have shown limited efficacy. Our studies have focused on the ability of melanomas to escape immune destruction by selective down-regulation of the very antigens that are being targeted with tumor vaccines, because even if a vaccine is able to raise highly specific cellular and humoral immunity, immunemediated destruction requires continued expression of target antigens. With melanoma vaccines, the melanocyte-specific antigens such as Melan-A/MART-1, gp100 and tyrosinase show extensive heterogeneity with respect to their expression levels in tumor cells. During our search for agents that can retain or enhance melanoma antigen expression, we showed that the MAP kinase pathway (MEK1/2) inhibitors PD98059 and U0126 were capable of enhancing expression of both mRNA and proteins for several antigens in melanoma cell lines. While treatment of melanomas with these agents induces dramatic morphological changes, and over time induces some apoptosis (approximately 25% at 4 days), there is a dose and time-dependent enhancement of antigen expression in viable cells. Data obtained with the MAP kinase inhibitors therefore suggested a possible link between B-raf function and antigen expression. However, we observed no simple relationship between B-raf mutational status and antigen expression levels in 23 melanoma cell lines. Furthermore, Western blotting showed no clear correlation between phospho-ERK levels, melanoma antigen expression, and B-raf mutational status.These data, together with MEK inhibitor enhancement of antigen expression, indicate that there are multiple influences on MAPkinase signaling that influence levels of antigen expression. Transient over-expression of B-raf offered a direct means of modulating MAP kinase signaling in a series of melanomas. Accordingly, we found that in four antigen-positive cell lines of variable V599E B-raf mutational status (two heterozygous wildtype /mutant, one homozygous wild type, and one homozygous mutant), transient over-expression of mutant B-raf resulted in increased ERK activation and reduction of Melan-A/MART-1 and gp100 antigen levels. We conclude that inhibition of ERK activation by MAP kinase pathway inhibitors promotes antigen expression in melanoma cell lines, irrespective of original antigen expression status (high, low or absent expression of Melan-A/ MART-1 and gp100). Conversely, when ERK activation is induced by transient constitutively active mutant B-raf transfection, antigen expression declines. These data indicate that while several factors may influence levels of antigen expression in melanomas, blocking MAP kinase activation enhances expression of several melanocyte-associated antigens, indicating that MAP kinase inhibition may prove helpful in targeting of melanomas in immunotherapy trials. Patients with advanced colorectal cancer have limited options for treatment. Immunotherapy, and especially dendritic cell (DC)-based vaccines, appears to be one of the most promising therapies. We identified peptides from CEA, MAGE and HER2/neu that stimulated CTL in vitro for further development. Previously, using normal healthy donors, we developed a large scale manufacturing process to produce adequate number of autologous DC loaded with the immunogenic peptides for use in a clinical trial. This process cultures mononuclear cells (MNC) in serum-free VacCellR media containing GM-CSF and IL-13. On day 7, DC enriched by elutriation were first pulsed with KLH, then matured with a bacterial membrane fraction from Klebsiella pneumoniae (FMKp) and IFNg, and pulsed with peptides. These matured DC were finally cryopreserved. This manufacturing process is capable of generating 10-21 vaccine doses per batch from a qualification study with normal healthy donors (n = 5). This process was further simplified by using the Gambro ELUTRAk Cell Separation System to perform the elutriation step with a single-use closed system sterile disposable set. Here, we present the qualification of this modified manufacturing procedure in three full scale Collidemk process runs. Three apheresis products from three normal healthy donors, after overnight shipping, contained 1.2 F 0.1  10 10 MNC with monocytes ranging from 13 to 18%. MNC were cultured for 7 days in VacCell media containing GM-CSF and IL-13. Enrichment by elutriation generated 1.1 F 0.3  10 9 DC, with a mean viability and purity of 96 F 2% and 93 F 4%, respectively. Upon maturation and peptide pulsing these DC showed typical patterns of phenotypic maturation, up regulation of CD25, CD80, CD86 and CD83 and down regulation of CD14 with secretion of IL-12p70 and TNFa. The process generated 7.9 F 3.0  10 8 viable DC that is equivalent to 23 F 8 vaccine doses per process. Based on these data, the process to manufacture the Collidem vaccine was amended to use this closed system elutriation method, and a Phase I / II clinical trial is underway to evaluate the Collidem peptide pulsed DC vaccine for treatment of colorectal cancer patients. Hfgl2 Prothrombinase/Fibroleukin Expression in Cancer and Its Potential Clinical Significance. 1 Objectives: Immune coagulation, microthrombus and fibrin deposition within the microvasculature are major contributors to the pathogenesis of xenograft rejection, viral induced hepatocellular injury and cytokine induced fetal loss syndrome. Here we investigated the contribution of the novel gene fibrinogen like protein 2 (fgl2) prothrombinase mediated immune coagulation in cancer and its potential clinical significance.Methods: Malignant tumor tissues were obtained from 11 patients with colon cancer, 15 patients with cervical cancer, 10 patients with breast cancer, 9 patients with esophageal cancer, 12 patients with lung cancer, 16 patients with gastric carcinoma. Immunohistochemistry and in situ hybridization were used to detect the hfgl2 prothrombinase and hfgl2 mRNA in tumor tissues. Antibodies for CD8, CD57, CD68 and vWF were also used in series histological sections to determine the cellular types in which the hfgl2 was expressed.Results: hfgl2 prothrombinase and hfgl2 mRNA was expressed in almost all the above cancers. Further more, it was evidenced that hfgl2 was highly expressed not just in cancer cells, but also in interstitial infiltrated cells include macrophages, NK cells, CD8+T lymphocytes and vascular endothelial cells.Conclusion: The expression of hfgl2 in cancer cells and activated interstitial infiltrated cells may contribute to the characteristic statue of hypercoagulability, which in turn induces tumor angiogenesis and metastasis. The molecule of hfgl2 may serve as a potential therapeutic target for the management of tumor development. This work was supported by the National Science Objective: Although the pathogenesis of primary central nervous system lymphoma (PCNSL) remains unclear, it is hypothesized that specific chemokine-chemokine receptor interactions contribute to localization of malignant B lymphocytes to the brain and eye. One candidate mediator is the lymphoid chemokine, stromal cell-derived factor-1 (SDF-1, CXCL12). Although initial work focused on its critical role in hematopoiesis, more recently the participation of SDF-1 in neural development has been recognized; SDF-1 is constitutively expressed by brain neurons and endothelium, neuroglia and meningeal cells. Consequently we studied the expression of this chemokine in PCNSL.Methods: Formalin-fixed, paraffin-embedded specimens from 10 patients with PCNSL were cut 3 microns in thickness and stained by standard indirect immunohistochemical methods, using a goat polyclonal anti-human anti-SDF-1 antibody (Santa Cruz Biotechnology) at a concentration of 5 Ag/mL. Following deparaffinization of the tissue, antigen retrieval was performed by boiling the sections for 10 minutes in 10 mM citrate buffer at pH 6.0. Normal tonsil, and astrocytoma and meningioma biopsies were also immunostained. Negative controls were prepared by substituting goat IgG (Sigma) for the specific antibody.Results: Positive staining for SDF-1 was identified in all 10 of the PCNSL biopsy specimens. This expression was localized to neurons, endothelial cells and meningeal cells. Weaker staining was also observed in lymphoma cells that were either diffusely distributed throughout the brain tissue or present as perivascular infiltrates. Neuronal and meningeal expression of SDF-1 was noted in the astrocytoma and meningioma biopsies; tonsil stained positively for SDF-1 in the crypt and outer epithelium, and within the tonsil proper. Interestingly, a mouse monoclonal anti-human SDF-1 antibody (R&D Systems) that recognized SDF-1 in tonsil showed no reactivity in either normal brain or PCNSL biopsy specimens.Conclusions: Expression of SDF-1 occurs within PCNSL lesions in the brain, as well as normal brain tissue. Phase 1-2 Evaluation of Different Immunotherapy Protocols for GBM. Glioblastoma Multiform (GBM) cells escape immune recognition by two major mechanisms. These cells produce strong anti-inflammatory substances in the local tumor micro environment and fail to express MHC II proteins as well as costimulatory and adhesion molecules on their surface. Mixed Leukocyte Culture Cytoimplant (MLC) has been previously shown to function as a powerful intratumor pro-inflammatory cytokine pump, which can reverse the anti-inflammatory activity of the tumor microenvironment (Cancer, 2000 (Cancer, , 88: 1325 (Cancer, -1335 . Tumor-B cell hybridoma vaccines (TBH) have been shown to function as antigen presenting cells, which can facilitate CD4 T cell recognition of the tumor antigen (Transfus Sci 1996, 17: 643-649) . The use of each of these therapies as single agents or combined have been tested successfully in clinical trials for others tumors (for pancreatic adenocarcinoma, ASCO Proceeding 2001, 20: 264a, and for breast cancer ASCO Proceeding 2003, 22: 182.) In this phase one study we have evaluated toxicity and possible antitumoral effect of each treatment and the combination of both. As a preliminary study 11 consecutive patients with GBM according to standard selection criteria were divided in three treatment groups. However, at the moment the Kaplan Meyer analysis does not show significant difference in the survival between the three groups. Treatment with MLC has a strong and rapid therapeutic effect, but it is limited in duration. It can generate sever encephalitis. Treatment with TBH has a slow and lasting therapeutic effect. It does not generate encephalitis. Treatment with MLC + TBH acts synergically, provoking a rapid, strong and lasting therapeutic response. In 50% of patients, it generated encephalitis. The three immunotherapy regimens seems to have strong antitumoral effect but they differ on velocity and the risk to develop secondary severe brain inflammation which is a complication that seems to be related with the MLC procedure and start around 15 days after the it was done. No other major toxic effect was observed. Suppression of Lck Sensitizes Acute Lymphoblastic T Cells to the Antiproliferative Action of Interferon Alpha.S. 1 1 Medicine, Stanford University, Stanford, CA, USA.We studied the effects of Lck suppression on the sensitivity of acute lymphoblastic T cells to the antiproliferative action of interferon alpha. Short interfering RNA (siRNA)-mediated Lck ablation reduced the activity of downstream signaling pathways indicating that Lck is constitutively active in lymphoblastic T cells despite the absence of T cell receptor stimulation. Lck-deficient and Lck siRNA-treated Jurkat cells were drastically more sensitive to the antiproliferative effect of interferon alpha than wildtype or untreated cells through a reduction in the rate of S-phase progression. Interestingly, Lck deficient cells were not more sensitive to the proapoptotic action of interferon alpha. Exposure of a panel of acute lymphoblastic T cells to an Lck-specific inhibitor similarly sensitized the cells to the antiproliferative effect of interferon alpha. This work provides a rational basis for the combined use of Lckspecific inhibitors and interferon alpha for the treatment of T cell acute lymphoblastic leukemia. We are currently employing reverse phase protein microarray technology to explore the mechanism by which Lck inhibition sensitizes interferon alpha activity. Regulatory Cells and Human Malignant Gliomas. Anderson, 1 J. N. Bruce, 1 D. E. Anderson. 2 We have isolated and analyzed both tumor cells and tumor infiltrating lymphocytes (TILs) present in ex vivo central nervous system (CNS) tumor specimens. We used flow cytometry to examine immunologically important cell surface molecules on glioblastoma multiformae (GBM) tumor cells prior to any culturing in vitro, and screened supernatants from ex vivo GBMs for secreted cytokines. Tumor cells expressed moderate to high levels of both HLA class I and class II molecules, suggesting that tumor cells could directly interact with and influence TILs. In addition, a majority of the GBMs examined (n = 10) secreted high amounts of the immunosuppressive cytokine IL-10. Based on these observa-tions, we hypothesize that GBM tumor cells promote the differentiation and/or expansion of IL-10-secreting type 1 T regulatory (Tr1) cells, which in turn suppress tumor immunity in situ. Consistent with this hypothesis, additional preliminary observations suggest that a CD25+ population of CD4+ TILs, which appear to represent effector cells activated in situ, infiltrates GBMs and that these cells are notable for their secretion of IL-10. These data suggest a novel mechanism by which the tumor microenvironment of GBMs influences the function of TILs and hinders their ability to mount an effective tumor response. Cancer is a disease that affects more than a million individuals every year. The pathogenesis of cancer is not yet completely understood and is considered to be multifactorial. Although there is a wide range of behavior between different cancers, all cancer cells present atypical morphology, bizarre mitotic cycles and uncontrolled growth rate, ultimately leading to invasion and killing of the host. The human immune system represents a powerful mechanism for detecting and destroying cancer cells. Immune recognition is mediated by the major histocompatibility complex class I (MHC I) molecules, which scan the cellTs proteome and carry small peptides of intracellular origin to the cell surface. Effector T-lymphocytes (CTL) survey MHCpeptide complexes and target cells displaying cancer-specific peptides. What epitopes differentiate the cancer cells from normal cells? Recognizing the MHC-peptide phenotype of cancer cells is a critical step in the development of CTL-activating vaccines and/or the design of new therapies. The process of epitope discovery from cancer cells requires large amounts of HLA to extract and identify the peptides presented by the MHC I. Therefore, our immediate goal is to transfect and express the soluble human MHC I molecules (sHLA)-A*0201 and -B*0702 in two or more cancer cell lines that will allow the production of large quantities of sHLA and facilitate the elucidation of MHC-epitopes presented on cancer cells. Materials and Methods. Breast cancer cell lines MCF-7 and MDA-MB-231 as well as lung cancer cell lines A549 and NCI-H292 were transfected with sHLA-A*0201 and sHLA-B*0702. sHLA constructs lack the intracellular and transmembrane domains of the a-chain, and were modified at the 5V end by adding the last 10 carboxi-terminal amino acids of the rat VLDL receptor (VLDLr). sHLA-VLDLr were cloned into the mammalian expression vector pcDNA3.1(-) Geneticin or Zeocin (Invitrogen) using FuGene 6 Transfection Reagent (Roche). Transfected cell lines were selected with the specific antibiotic, and assayed for production of the transfected A*0201 and B*0702 molecules using an in-house ELISA. Several positive clones were obtained form each cell line and high producers were selected by sequential subcloning and ELISA testing for sHLA protein production. Two breast cancer and two lung cancer cell lines were successfully transfected with sHLA-A*0201 and sHLA-B*0702. Stable clones producing sHLA z50 ng/ml are now available for the production of high quantities of sHLA and the future detection of unique peptide epitopes that distinguish cancer cells from normal cells. Stable and high sHLA-producer cancer cell lines are an important preliminary step in the cancer epitope discovery process. Fulfillment of our immediate goal enables us with the tool to proceed in the discovery of unique epitopes that characterize the MHCpeptide phenotype of cancer cells.Sa2.125. Serologic Study Using a GST-Capture ELISA for Determination of Anti-HPV16 Antibodies in Tunisian Women.M. 1 Biology, Faculty of Sciences, Bizerte, Zarzouna, Tunisia; 2 DKFZ, Heidelberg, Germany; 3 Hospital la Rabta, Tunis, Tunisia; 4 Institute Salah Azaiz, Tunis, Tunisia; 5 Hospital la Rabta, Tunis, Tunisia; 6 DKFZ, Heidelberg, Germany; 7 Biology, Faculty of Sciences, Bizerte, Zarzouna, Tunisia.Cervical cancer is the second most tumor in tunisian women just after breast cancer.Human papillomavirus (HPV) infection is considered the main cause of invasive cervical cancer espacially HPV16 and HPV18; Moreover, antibodies against the E6 and E7 proteins of HPV types 16 and 18 have been found to be strongly associated with cervical cancer. Many assays can be used to define presence of these antibodies but the majority of them have low sensitivity.The present work is the first one in Tunisia in which we have used a GST-capture ELISA in order to estimate the antibody response against HPV16 E6 and HPV16 E7. These antigens were overexpressed in E.coli as GST fusion proteins as described by the research group of Pawlita.Using this system, we have analysed 205 sera; 71 cases of cervical cancers, 64 cases of inflammation of the cervix and 70 controls. Results showed that among cancers, positivity is 37% and 42% for 16 E6 and 16 E7 respectively and it is 0% and 2% for inflammations, 3% in controls for both 16 E6 and 16 E7. Ipsilateral Lymphadenectomy To Inhibit Corneal Allograft Rejection in Rats. 1 Ocular Surface Center, Sun Yat-sen University, Guangzhou, Guangdong, China; 2 Ophthalmology of Tongji Medical College, Huazhong University of Science and Technology.Objective: To investigate use of ipsilateral lymphadenectomy therapy for inhibiting immune rejections in rat corneal transplantation. Methods: Corneal allogenic transplantation models were established in rats. 18 female Wister rats were used as donors,and 36 female sprague dawley (SD) rats were used as recipients. After corneal penetrating transplantation, recipients were randomly divided into 3 groups: Group A was the control group; Group B, the bilateral lymphadenectomy group; Group C, the ipsilateral lymphadenectomy group. Among of 12 rats in each group, the corneas of 2 rats in each group were used for pathological study at day 14 after the transplantation, the other 10 rats were used for studying corneal immune rejection with a slit lamp. The point in time when allograft rejection occurred was recorded and mean survival time (MST) were compared. In addition, the rejection index, clarity, edema and vascularization of the grafts were examined and compared 14 days after transplantation. Results: MST of the grafts in group B and C were (46.30 F 9.464)days, (44.43 F 7.604)days, respectively, and were increased significantly ( P b 0.01) in comparison with the group A, which had MST of (10.71 F 1.567) days. The difference in the MSTs between group B and C was not significantly (P N 0.05). Pathological study showed that at 14 days after transplantation, the corneas in group A were in a state of edema and showed disturbed collagen fibers and very significant neovascularity.However, for corneas in group B and C, no significant changes were detected compared with nomal corneas except for a reduced density of endothelial cell. 14 days after corneal transplantation.The rejection index for group B and C,was much lower than that for group C. Conclusions: Both bilateral and ipsilateral lymphadenectomy therapies have a significant effect in preventing corneal allograft rejection. Ipsilateral lymphadenectomy is a less complex surgical procedure and is just as effective in preventing rejection. The exact cause of glaucoma is not known, however in the recent years it has been suggested that endothelin-1 (ET-1) on aqueous humor (AH) is related with the generation of glaucoma in humans and animals models.In order to understand the participation of ET-1 in the maintenance of the different types of glaucoma, we determined the ET-1 concentration in AH and plasma from glaucoma patients.Materials and Methods: After informed consent, blood and AH samples were taken from patients with glaucoma treated with trabeculectomy (Primary open-angle glaucoma-POAG-, Acute closed-angle glaucoma-ACAG-, Pseudoexfoliative glaucoma-PG-) and from patients with senile cataract treated with phacoemulsification and without antecedent of glaucoma (Control group, CG). ET-1 determination was made by a chemiluminescent immunoassay and the results were analyzed with the Mann-Whitney Rank Sum Test considering a difference statistically significant with a P b 0.05.Results: ET-1 concentration in AH from glaucoma patients was 3.9 F 0.9 pg/100ml, while in CG group was 5.2 F 1.5pg/100ml; ET-1 plasma concentration from glaucoma patients was 0.8 F 0.1 pg/100ml, while in CG group was 0.4 F 0.1 pg/100ml (P = 0.008). We do not find statistical differences in the concentration of ET-1 in AH between the different groups of glaucoma patients: POAG 3.2 F 0.8 pg/100ml, ACAG 7.9 F 3.1 pg/100ml, PG 3.2 F 2 pg/ 100ml, and we do not find statistical differences when we compared each group with CG group. Also, we do not find statistical differences in the concentration of ET-1 in plasma samples between the different groups of glaucoma patients: POAG 0.9 F 0.2 pg/100ml, ACAG 0.6 F 0.2 pg/100ml, PG 0.7 F 0.2 pg/ 100ml. However, when we compared ET-1 concentration in plasma from each group of glaucoma patients with the CG group, we found that only in the POAG patients a difference statistically significant (P = 0.014).Conclusions: Our results suggest that only in the groups of patients with POAG could be and association with the ET-1 plasma concentration and glaucoma. We consider that the diverse clinical presentations of glaucoma studied here are a consequence of a different molecular process, in which of them, the ET-1 could play a minimum role. At the present time, there are developing drugs against endothelin to treat glaucoma, however the differences observed in our study should be considerer in order to guarantee the success of new therapies for glaucoma. LPS Stimulation Induce an up Regulation of TLR-4 on Limbal Epithelial Cells without Secretion of TNF-Alpha. 1 1 Research Unit, Institute of Ophthalmology, Conde de Valenciana, Mexico, D.F., Mexico, D.F., Mexico.Introduction: Little is known about the expression of natural immune receptors as toll like receptors in corneal epithelium, and their function in ocular immune response is controversial.Objective: We sought to determine the extracellular expression of TLR-4 on human limbal epithelial cells cultivated in vitro and if so, to determine its cellular function after LPS stimulation.Matherials and Methods: From sclera-corneal rims, limbal epithelial cells were isolated and grown in the presence of supplemented hormonal epithelial medium at 378C and 5% CO2 until confluence. At passages one or two, the cells were exposed to different doses of LPS from E. coli for 24 h. After stimulation, the cells were recovered and stained with PE-conjugated monoclonal antibodies against human TLR-4 and analyzed by flow citometry; mRNA was obtained and RT-PCR was performed for the identification of TLR-4, GADPH was used as an internal control. Secretion of TNF-alpha by these cells was evaluated by ELISA on the supernatant. PBMC were used as LPS activation controls.Results: Limbal epithelial cells expanded in vitro expressed constitutively low density TLR-4; after stimulation with LPS the expression of TLR4 was augmented taking into account the medium fluorescence intensity. A similar behavior was observed at the mRNA level, the expression was augmented after stimulus. When TNFalpha was evaluated, interestingly, this cytokine was not detectable at any concentration of LPS and even at 48 h of stimulus. PBMC secreted optimal concentrations of TNF-alpha after LPS stimulation.Conclusions: Although the extra cellular expression of TLR4 on limbal epithelium stimulated in vitro up-regulates TLR4, its function seems not to be associated with the secretion of TNFalpha on limbal epithelium.Sa2.130. CD80 and CD86 Expression on Corneal Epithelial Cells Infected with Adenovirus.MaCarmen Jimenez, 1 Herlinda Mejia, 1 Marisela Linares, 1 Alejandra Sanchez-Navarro, 1 Raul Suarez, 1 Yonathan Garfias. 1 1 Research Unit, Institute of Ophthalmology, Fundacion Conde de Valenciana, Mexico, D.F., Mexico, D.F., Mexico.Introduction: CD80 and CD86 belongs a family of proteins named B7. B7 molecules costimulate T cell during immune activation. Normally, the corneal epithelial cells do not have any expression of those molecules on their surface. The objective of this study is to determine if the corneal epithelial cells induce B7molecules after a viral infection.Materials and Methods: Corneal epithelial cells were isolated from human corneas treated with dispase-II, and grown in the presence of supplemented hormonal epithelial medium at 378C and 5% CO 2 until confluence. At passages one or two, the cells were exposed to different doses of adenovirus 5 (Ad5) for 2 h. After infection the cells were washed three times and then cultured at different times. Then, the cells were recovered and stained with PE-conjugated monoclonal antibodies (mAbs)against human CD80 and CD86 and with FITC-conjugated mAbs against human cytokeratin and the results were analyzed by flow citometry.Results: Non infected corneal epithelial cells did not express at any time CD80 or CD86; however Ad5 infected epithelial cells were positive to CD80 since 24h (40%) raising its maximum level at 72 h (~90%), CD86 expression on epithelial corneal cells was detected also at 24h (20%) raising its maximum level at 72h (~70%). Conclusions: Our results suggest that corneal epithelial cells express CD80 and CD86 after virus infection.Sa2.131. I. Kump, 1 K. L. Moeller, 1 S. Kurup, 1 G. F. Reed, 1 R. B. Nussenblatt. 1 1 Laboratory of Immunology, National Eye Institute, NIH, Bethesda, MD, USA.Purpose: To analyze differences in response to treatment of ocular Adamantiades-BehcetTs disease (ABD) in the 1970Ts, 1980Ts and 1990Ts.Methods: Medical records of thirty six patients with uveitis due to Adamantiades-BehcetTs disease followed at the National Eye Institute (NEI) were reviewed.Results: All the patients were divided in 3 groups according to the time of follow-up: first group was followed from 1962 untill 1972, second group from 1983 untill 1992 and the third group from 1992 through 2004. Visual acuities and degrees of inflammation in each group were recorded at each visit. There were 12 patients (24 affected eyes) in the 1970Ts group, 7 patients (14 eyes) in the 1980Ts group and 17 patients (34 eyes) in the most recent group. Therapeutic agents used in the 1970Ts group included systemic steroids, methotrexate, 6-mercaptopurine, azathioprine, cyclophosphamide and chlorambucil. Agents used in the 1980Ts group included systemic steroids, cyclosporine, chlorambucil and colchicine. Medications used in the most recent group included systemic steroids, cyclosporine, azathioprine, daclizumab, mycophenolate mofetil, methotrexate and infliximab. Statistical analysis showed that average logMAR change per year was 0.6479 in the 1970Ts group, -0.0225 in 1980Ts group and -0.0272 in the most recent group.Conclusion: Adamantiades-BehcetTs disease is a severe blinding disorder. Larger studies may be more empowered to demonstrate the trend for improvement due to introduction of newer agents and directed therapy. Purpose: To analyse visual outcomes in children with juvenile idiopathic arthritis-associated uveitis.Methods: Charts of 89 children with JIA-associated uveitis were reviewed.Results: Among 269 children with uveitic syndromes referred to a tertiary eye center 89 children (33%) had juvenile idiopathic arthritis associated uveitis. The process was bilateral in 76 children. Seventy three patients were females, 84% of patients were Caucasian. The patients with JIA-associated uveitis developed numerous complications in the course of their disease: among 165 affected eyes 105(64%) developed cataracts, 33 (20%) developed increased intraocular pressure, 76 (46%) eyes developed band keratopathy, posterior synechiae were present in 96 (58%) eyes. Among 89 children 73% were on immunomodulators, and 40% were treated with non-steroidal anti-inflammtory agents alone or in combinaion with immunomodulators, and 21% were treated with topical and or/systemic steroids. Among 65 children who required immunomodulation, only one chemotherapeutic agent was used in 30 children, two agents in 21 children, and three or more in 14 children. Visual acuities of the patients were documented and compared at standard intervals. By mixed models linear regression, visual acuity improved an average of 0.0113 logMAR units at each two-month visit (P = 0.032).Conclusion: In spite of the severity of JIA-associated uveitis, much of the childrenTs vision can be preserved if the patients are treated appropriately. Increased Expression of IKBA Permits Tolerance Induction by TGFB-Treated Antigen Presenting Cells. Antigens introduced in the anterior chamber of the eye are processed and presented by resident antigen presenting cells (APCs) in a manner that results into antigen-specific peripheral tolerance that is characterized by suppression of Th1-mediated immune response such as delayed type hypersensitivity (DTH). Similarly, conventional APCs such as peritoneal exudates cells (PECs) or a macrophage hybridoma clone (#59) that are treated with TGFh in vitro can induce comparable peripheral tolerance. Such TGFhtreated APCs are known to express a unique set of genes that are essential for their tolerance inducing ability. Suppression of two genes regulated by NFnB, IL-12 and CD40, is critical for their tolerizing property. The InB proteins that prevent nuclear translocation of NFnB dimers therefore, can also support suppressed expression of IL-12 and CD40. Antigen presenting cells treated with TGFh, were found to up-regulate their expression of InBa. In this series of experiments we have assessed contribution of InBa to tolerance promoting properties of APCs. Macrophage hybridoma stably expressing anti-InBa siRNA or ectopically overexpressing InBa were examined for their ability to express InBa protein (using western blot), suppress OVA-specific DTH and secrete IL-12 in culture supernatants. These APCs were pulsed with ovalbumin (OVA) and cultured overnight in the presence or absence of TGFh2 (5 ng/ml). Increased levels of InBa protein were detectable in TGFh-treated #59 and InBa overexpressing #59, however APCs expressing anti-InBa siRNA failed to do so. Inhibtion of IL-12 secretion by TGFh treatment was found reversed in APCs expressing anti-InBa siRNA. While both TGFh-treated #59 and #59 overexpressing InBa suppressed OVA-specific DTH, such suppression was not induced by TGFh-treated #59 expressing anti-InBa siRNA. Nuclear p50 levels were significantly decreased in TGFh-treated #59 and InBa overexpressing APCs, while such a decrease was prevented in TGFh-treated anti-InBa siRNA expressing #59. Such increased expression of InBa permits tolerance induction by these APCs.Sa2.134. CD36 and Thrombospondin Interaction Is Essential for the Tolerance Inducing Properties of TGFB-Treated Antigen Presenting Cells. Antigen presenting cells (APCs) in the anterior chamber of the eye are known to induce antigen-specific systemic tolerance in which Th1-mediated immune response such as delayed type hypersensitivity (DTH) is suppressed. Active TGFh, abundantly available in the ocular environment, confers such toleranceinducing property on these resident APCs. Similarly, in vitro treatment of conventional APCs with TGFh converts their functional phenotype to that of tolerizing APCs. Investigation of molecular mechanisms underlying this conversion has revealed significant involvement of various molecules such as TNFa, MIP-2, TGFh2, CD40 and IL-12. Products of these genes, via various pathways contribute to systemic tolerance induced by TGFhtreated APCs. More recently, we detected increased expression of extracelluar matrix protein, thrombospondin (TSP), in TGFhtreated APCs. In this report we have assessed molecular interactions of TSP that lead to tolerogenic phenotype of these APCs. Conventional APCs (either hybridoma cell line #59) or thioglycollate-elicited peritoneal exudates cells (PECs) can express TSP receptors such as CD47 as well as CD36. Macrophage hybridoma #59 or peritoneal exudates cells collected from wild type(WT) or TSP-1 null or CD36 KO mice were used as APCs. These APCs, pulsed with ovalbumin (OVA) and treated with TGFh2 (5 ng/ml), were examined for their ability (1) to suppress OVA-specific DTH response, (2) to inhibit IL-12 secretion by ELISA and RT-PCR and (3) to enhance TSP or TNFa expression by RT-PCR. While WT APCs treated with TGFh or #59 treated with TSP suppressed OVA-specific DTH, both TSP-1 and CD36 deficient APCs failed to do so in response to TGFh treatment. In case of TSP-1 deficient APCs, exogenously added TSP restored their ability to acquire tolerogenic properties upon TGFh exposure. These results support the possibility that TSP expressed by TGFh-treated APCs contributes significantly to tolerance inducing property by facilitating inhibition of IL-12 secretion and expression of TNFa. Interaction of TSP with its receptor CD36 is essential for the tolerizing phenotype of TGFh-treated APCs. 1 7 IL-10, an anti-inflammatory cytokine, has been shown to exhibit stimulatory functions including CD14 upregulation on human monocytic cells. CD14-mediated signaling following LPS stimulation of monocytic cells results in the synthesis of proinflammatory cytokines. Our flow cytometry results show that LPS-induced CD14 expression on monocytic cells may be mediated by endogenously produced IL-10. To investigate the molecular mechanism by which IL-10 enhances CD14 expression, both human monocytes and the promyelocytic HL-60 cells were used as model systems. IL-10 induced the phosphorylation of phosphatidylinositol 3-kinase (PI3K) and p42/44 extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPK) as determined by western blotting with phospho-specific anitbodies. By employing specific inhibitors for PI3K (LY294002) and ERK MAPKs (PD98059), we demonstrate that LY294002 either alone or in conjunction with PD98059 inhibited IL-10-induced phosphorylation of signal transducer and activator of transcription-1 (STAT-1) and consequently CD14 expression. However, IL-10-induced STAT-3 phosphorylation remained unaffected under these conditions. Furthermore, LY294002 and PD98059 inhibited the binding of the STAT-1 transcription factor to its binding site in the CD14 promoter. Finally, transfection of HL-60 cells with STAT-1 interfering RNA vectors inhibited IL-10-induced CD14 expression. Taken together, these results suggest that IL-10-induced CD14 upregulation in human monocytic cells may be mediated by STAT-1 activation through the activation of PI3K either alone or in concert with the ERK MAPK. Altered Cytokine Production and Reduced Membrane Signaling through the Antigen Receptor in CD4+ T Cells Overexpressing Ly-6A.2 Molecule.Anil Bamezai, Jennifer Reed, Abraham Chacko. 1 Biology Department, Villanova University, Villanova, PA, USA.Mouse Ly-6 molecules serve as excellent differentiation markers on immune cells of hematopoietic origin, but their influence on cellular differentiation is unknown. CD4+ cells over-expressing Ly-6A.2 resist polarization to either Th1 or Th2 phenotype. To understand the mechanism underlying the polarization tolerance in Ly-6A.2 over-expressing CD4+ T cells, we examined their cytokine profile in response to varied antigenic stimulation under primary culture conditions. Ly-6A.2 over-expressing cells generated higher levels of both Th1 and Th2 signature cytokines in a dose dependent manner. In addition, the CD4+ T cells over-expressing Ly-6A.2 showed reduced phosphorylation of LAT than the controls. Our results suggest that Ly-6A.2 expression on CD4+ T cells diminishes their membrane proximal signaling in response to the antigen receptor and therefore might contribute to their altered cytokine profile. These results also suggest that CD4+ T cells capable of producing high levels of Th1 and Th2 signature cytokines in response to antigen stimulation are resistant to polarization to either Th1 or Th2 phenotype. We hypothesized that proatherogenic T cells are controlled by cytokines network balance. Among them, TGF-b has been implied in atherogenesis, but its mechanism of action remains unclear. Taken together, abrogation of TGF-b signaling in T cells able to accelerate the atherosclerosis and permit to suggest that TGF-b reduces atherosclerosis by dampening T cell activation. Inhibition of T cell activation may therefore represent a strategy for antiatherosclerotic therapy. Goal: to analyze the possible evidence for TGF-b action on lymphocytes functions (G o chromatin topography changes) in vitro at cerebral and coronary atherosclerosis. The lymphocytes chromatin nuclei behavior was estimated after 30, 60 and 360 min for each samples. The chromatin of lymhocytes nuclei was studied using Computer TV Morphodensitometry System bDiaMorphQ (Russia) in the smears dyed especially for DNA. Results are contraindicated to habitual opinions about common atherogenic mechanisms without evaluation of organtarget influence. PDGF at cerebral atheroslerosis acted in other manner or in assembly to other factor, which are could be known). However, more complex manner of chromatin changes of lymphocytes, implicated in atherogenesis, have shown, that they have underwent by most of involved components of cytokines network factors, which are reversed timely. The main detected features at cerebral in comparison to coronary atherosclerosis have consist of initial hetero-and euchromatin rebuilding. Obtained results have conflicted to becoming fixed opinion that cerebral and coronary atherosclerosis are common process, unified any atherosclerosis without locations evaluation. So it is possible, that the detected changes have resulted more disseminated vascular implication in cerebral atherosclerosis in comparison to coronary ones. Obtained results are controversial to described the dampening lymphocytes activation, induced by TGF-b. We have no satisfied results, evidenced the TGF-b suppressive manner on lymphocytes functioning at atherosclerosis. Received results, in our opinion, reflects the more complicated interaction between TGF-b and lymphocytes functioning changes. It has been confirmed by the facts, that G 0 lymphocytes chromatin (dependent from concentration and exposition time) have changed its portrays on biphasic manner after PDGF-AB adding: initially at short period of time we detected the same nuclear activation (and gene expression activation, correspondingly) with following stable chromatin topography changes, reflected the stable lymphocytes functioning changes on pathologic manner, which is needed to more detail investigation. Live Measurement of Chemokine Triggered Integrin Activation on a Single-Molecule Level.R. 1 1 Institute of Pathology, Technical University of Munich, Munich, Germany.Atomic force microscopy (AFM) based force spectroscopy can be used for direct measurement and quantification of cell adhesion forces on living cells down to single-molecule interactions. This technique is used here to study the physiologic activation of alpha(4) integrins by the chemokine CXCL12 on living cells expressing the chemokine receptor CXCR4. At low physiologic concentration of CXCL12 the rupture force of a single pair of adhesion receptors increases from 40pN to 60-80pN, whereas at higher concentrations of CXCL12 it lowers the rupture forces back to 40pN. This study confirms that arrest chemokines such as CXCL12 can rapidly modulate the affinity of an integrin receptor. To date, this is the first direct measurement of chemokine induced affinity modulation of a single (adhesion) receptor as well as of the desensitization of a chemokine receptor, both measured on a molecular level on a living cell. AFM based force spectroscopy has evolved into a completely new pharmacological test for singlemolecule interactions on living cells. This technique will be used to elucidate the mechanisms involved in both physiologic leukocyte homing as well as organ-specific tumor cell metastasis. REFERENCES: Eibl RH, Atomic force microscopy measurement of SDF-1 mediated affinity modulation of single VLA-4-VCAM-1 bonds, In: Immunology 2004-Cytokine Network, Regulatory Cells, Signaling, and Apoptosis, 115-119; 2004, Medimond, Milano, Italy, ISBN 88-7587-069-1. Eibl RH and Benoit M, Molecular resolution of cell adhesion forces. IEE Proceedings-Nanobiotechnology 151 (3) TGF-beta1 is a multifunctional cytokine involved in several immunoregulatory processes and plays a critical role in the escape of some cancer from host immunity. HPV is the main etiologic agent in cervical cancer, and E6 and E7 oncoproteins have properties of transforming cell and transregulation of cellular genes. However, the mechanisms by which HPV induce TGF-beta1 in cervical cancer remain unclear. In this study we analyzed whether HPV-16 E6 and E7 oncoproteins are involved in the molecular mechanisms of TGF-beta1 gene expression. For that end, we amplified the human 5V-TGF-beta1 promoter by PCR from peripheral blood lymphocyte DNA, which was cloned in pBluescript and sequenced. This DNA fragment was subcloned in pBLCAT3 and several constructs generated by deletion of TGF-beta1 promoter. C33A cells were transfected with this constructs and evaluated the effect of HPV-16 E6 and E7 oncoproteins by cotransfection with pSV2E6 and pSV2E7. We observed that E6 and E7 oncoproteins induced two-fold promoter activity inside TGF-beta1 core promoter, while had not significant effect in other TGF-beta1 regulatory regions. We also observed several interactions DNA-protein between HPV-16 E6 and E7 oncoproteins with Sp1 regulatory elements of TGF-beta-1 core promoter by Footprinting and EMSA. In addition, we identified a super shift DNA-protein complex specific associated to Sp1 binding site of TGF-beta-1 core promoter when the anti-E6 or anti-E7 antibodies were used. The results suggest specific interactions between viral transcriptional factors inside of TGF-beta1 core promoter. These results may explain the molecular mechanisms of TGF-beta1 gene regulation in cervical cancer as well as tumor escape mechanisms from host antitumoral immune response. BACKGROUND: Orexin-A, also named hypocretin-1, is a novel neuropeptide secreted by specific neurons in lateral hypothalamus. Recent findings suggest that orexin-A provides a critical link between the peripheral energy balance and central nervous system mechanisms that coordinate sleep-wakefulness and motivated behaviors, mainly promoting food intake and suppressing energy expenditure. As orexin-A is an active mediator closely related with energy metabolism, we hypothesize that orexin-A may undergo a fluctuation during the severe metabolic impediment of acute inflammation, such as intestinal I/R injury.MATERIALS AND METHODS: An intestinal ischemiareperfusion (I/R) injury model of rats was established, and rats were divided randomly into six groups: sham-operation group, 60 min ischemia/30 min reperfusion group (I60VR30V), I60VR90V, I60VR150V, I60VR240V and I60VR360V, 9 rats each group. A highly sensitive orexin-A radioimmunoassay was used to check the change of orexin-A concentrations in plasma and hypothalamus tissue, and RT-PCR was used to detect the change of orexin-A mRNA expression in hypothalamus tissue. Therefore, the change of orexin-A levels both in peripheral blood and central secretory tissues before and after intestinal I/R injury could be investigated.RESULTS: Compared with before injury, plasma orexin-A levels of each group showed no significant difference. Compared with sham group after injury, both plasma and hypothalamus orexin-A levels of every other group showed no significant difference. Compared with sham group after injury, orexin-A mRNA expression of I60VR30V and I60VR90V decreased step by step, that of I60VR150V reached the lowest, and that of I60VR240V and I60VR360V recovered gradually to the level of sham group.CONCLUSION: Orexin-A has a delayed response to acute inflammatory stimuli such as intestinal I/R injury, and it may participate in metabolic disorders in the injury as inflammatory cytokines.Keywords: Ischemia-reperfusion, intestinal; Orexin-A; Radioimmunoassay; PCR; Inflammation, acute; Cytokine Su1.07. Increased Interleukin-6 Serum Levels in Subjects with Metabolic Syndrome. An activation of generalized non-specific inflammation could play a role in the pathogenesis of metabolic syndrome and its components. However, the data regarding the changes of proinflammatory interleukins in subjects with metabolic syndrome remain controversial. Therefore, the aim of this study was to investigate the levels of interleukin-6 in the serum in subjects with metabolic syndrome compared to those without this syndrome. The diagnosis of metabolic syndrome was made based on ATP-III criteria (2001). In the group of patients with metabolic syndrome there were 18 subjects with type 2 diabetes mellitus, 16 persons with impaired glucose tolerance, arterial hypertension was diagnosed in 45 subjects. In those persons without metabolic syndrome type 2 diabetes mellitus was registered in 3 subjects, impaired glucose tolerance was found in 7 persons, arterial hypertension was diagnosed in 8 subjects. All persons studied did not receive any medications for the treatment of diabetes mellitus or arterial hypertension at the time of the examination. There were no clinical signs of any concomitant disease in patients studied. The levels of interleukin-6 were measured by specific assay. We found that the levels of interleukin-6 were significantly increased in patients with meta-bolic syndrome compared to those without this syndrome-1.44 F 0.3 and 0.625 F 0.16 pg/ml, respectively, P b 0.05. We can conclude that elevated serum interleukin-6 levels could reflect an activation of generalized inflammation in patients with metabolic syndrome, which could contribute to the development and progression of atherosclerosis and high risk of cardiovascular disease in these patients. LIGHT Regulates CD86 Expression on Dendritic Cells through NF-kB, but Neither p44/42 MAPK nor JNK/AP-1 Signal Transduction Pathway.G. 2 1 Bone Marrow Transplantation, Rush University Medical Center, Chicago, IL, USA; 2 Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL, USA.The members of the tumor necrosis factor (TNF) family play pivotal roles in the regulation of the immune system. LIGHT is a type II transmembrane protein belonging to the TNF family that was originally identified as a weak inducer of apoptosis. This cytokine has been extensively studied on its role in T cell regulation. Recently, we identified its role in inducing maturation of dendritic cells, such as LIGHT upregulated CD86 expression on dendritic cells in our previous report. However, the signal transduction pathway on this regulation remain unknown. LIGHT upregulates CD86 expression on DCs through activation of NF-kB, but not p44/ 42 signal pathway, because inhibition of NF-kB activity by its inhibitor could blunt the effect of LIGHT in up-regulation of CD86 expression, but neither inhibitor of p44/42 nor JNK inhibitor has this effect. Thus we demonstrate that LIGHT regulates CD86 expression through NF-kB signal transduction pathway but neither p44/42 MPAK nor JNK/AP-1 signaling pathway. Histamine is a well known mediator of allergic inflammation, synthesized by one step enzymic reaction with histidine decarboxylase (HDC). The increase in vascular permeability, vasodilatation and the stimulation of nerve terminals of primary sensory neurons through the stimulationof H1-receptors conntribite to the facilitation of the inflammatory response. In addition to H1-receptor-mediated effects, histamine has been demonstrated to be involved in the regulation of innate and aquired immune responses through H2-receptors. Histamine also inhibited the ICAM-1 expression on monocytes in a human mixed lymphocyte reaction in the presence of IL-18. These effects of histamine were all mediated by H-2-receptors.Fulminant hepatic failure is pathologically characterized to be diffuse intrahepatic infiltration by inflammatory cells with massive multilobular necrosis. Heat-killed Propionibacterium acnes (P.acnes) followed by challenge with a low dose of lipopolysaccharide (LPS) induces acute and massive liver injury, mimicking fulminant hepatic failure. In the present study, we examined a functional role of inducible histamine in the protection against hepatic injury and lethality in P.acnes-primed and LPS-induced hepatitis, using HDC knockout and H2receptor knockout mice. Moreover, we investigated the effects of the inhibitor of histamine N-methyltransferase(HMT) on hepatitis. LPS challenge after P.acnes-priming increased HDC activity in the liver of wild-type mice, associated with a marked elevation of histamine and tele-methylhistamine levels. Western blotting showed the increase in 74 KDa HDC band in the liver. These results strongly indicated that HDC protein was induced by LPS in the liver and that the histamine produced had high turnover rate. HDC-like immunoreactivity was observed in CD68-positive Kupffer cells/macrophages. Treatment of wildtype mice with famotidine and ranitidine but not chlorpheniramine augmented hepatic injury and inhibited the survival rate. The same dose of P.acnes and LPS induced severe hepatitis and high lethality in HDC and H2-receptor knockout mice, the former were rescued by the subcutaneous injection of histamine. Histamine suppressed the expression of IL-18 and TNF-a in the liver, leading to the reduced plasma levels of inflammatory cytokines. HMT inhibitor had similar effects to histamine. These findings indicated that endogenously produced histamine plays a very important role in preventing excessive innate immune response in endotoxin-induced fulminant hepatitis through the stimulation of H2-receptors. VEGF is considered to be one of the most important angiogenic factors. It is known to be chemoattractive for human monocytes and to possess some immunoregulatory influence on T-cells. The aim of the study was to evaluate the mRNA expression of VEGF receptors: VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1) in murine immunocompetent cells. Isolated thymocytes, lymph node cells, whole thymus and peritoneal macrophages from intact C3HA mice were used for investigation. Peritoneal cells were allowed to adhere for 2 hours and then washed from non-adherent cells. The level of mRNA expression was studied by reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was isolated with the help of guanidine thiocyanate method. Synthesis of cDNA templates was carried out using 2 Ag of total RNA, oligo d(T)15 primers and M-MLV reverse transcriptase (Promega) according to the manufacturerTs instructions. Specific primers used for PCR amplifications were: VEGFR1 forward primer-5V-GAAGCGGTT-CACCTGGACTGAGACC-3V; VEGFR1 reverse primer-5V-GGCQ TTTGCTGGGGGGATTTCTCTAA-3V; PCR product size 432 bp; VEGFR2 forward primer-5V-ACAGACAGTGGGATGGTC-CTTGCAT-3V; VEGFR2 reverse primer-5V-AAACAGGAGGT-GAGCTGCAGTGTGG-3V; PCR product size 272 bp. Primer pairs were designed from nucleotide sequences available in public database. As a control for housekeeping gene an RT-PCR procedure for h-actin was also delivered. Whole thymus expressed mRNA for both VEGFR1 and VEGFR2, while isolated thymocytes expressed only VEGFR2 mRNA. Peritoneal macrophages as well as isolated lymph node cells expressed mRNA for both receptors. Incubation of peritoneal macrophages in the presence of 50 ng/ml of VEGF during 24 h resulted in the enhancement of VEGFR1 mRNA expression. This suggests that VEGF may directly influence thymic cells and have some specific influence on the development of T-cells. As it was previously shown VEGF can modulate thymocyte mitogen-induced proliferation and spontaneous apoptosis in vitro and evokes thymic involution while administered in vivo (Ohm et al., 2003) . The expression of VEGFR2 mRNA in murine peritoneal macrophages is also a novel finding. These data indicate that tissue macrophages possess both receptors unlike blood monocytes that are known to express only VEGFR1. Rationale: Polymorphonuclear neutrophils (PMN) are major players in inflammation. They are the first cells to arrive at an inflammatory site and are involved in the inflammatory response. Inflammation is largely resolved when PMN undergo apoptosis and are then ingested by phagocytes such as macrophages. Many cytokines can modulate PMN functions including apoptotic rate and the ability to exert phagocytosis. IL-4 is particularly known to enhance PMN phagocytosis and to delay apoptosis. After binding to their specific receptor, many cytokines activate the intracellular Jak/ STAT pathway. It was recently discovered that the Jak/STAT pathway can be regulated by a family of proteins named SOCS (suppressor of cytokine signalling), that inhibit the pathway via a feedback loop mechanism. The aim of the present study was to evaluate the expression of SOCS and their modulation in PMN and in the PLB-985 cell line following cytokine stimulation. PLB-985 are immature promyelocytic cells that acquire a neutrophil-like phenotype when dimethylsulfoxide (DMSO) is added to the culture medium. Methods: PMN were freshly isolated from the venous blood of healthy volunteers. Differentiated PLB-985 (PLB-985D) were obtained after incubation of PLB-985 cells with 1,25% DMSO for 6 days. Cells were incubated with either G-CSF, GM-CSF, IL-2, IL-4 or IL-6 for 1 to 8h. RT-PCR was used to detect SOCS mRNA and protein expression was studied by immunoblotting using specific antibodies. The proteasome inhibitor MG132 was used to investigate the SOCS protein pool that is rapidly degraded in the cell. Cycloheximide (CHX) was used to inhibit protein synthesis and to answer whether or not SOCS proteins are de novo synthesized. Results: G-CSF, GM-CSF and IL-4 were found to induce the mRNA expression of SOCS3 in PMN after 1h of stimulation. PMN, but not PLB-985D cells, were found to express a basal level of the SOCS3 protein. After pre-incubation with MG132, treatment of cells with G-CSF, GM-CSF or IL-4 enhanced the level of SOCS3 in PMN and induced its expression in PLB-985D at the protein level. CHX reversed the increase of SOCS3 in PMN following stimulation with cytokines. CIS and SOCS2 mRNA expressions varied in PMN and PLB-985D, but this was not observed at the protein level. Conclusions: SOCS3 mRNA and protein expression increased in PMN and PLB-985D after stimulation with G-CSF, GM-CSF or IL-4. SOCS3 is rapidly degraded in PMN and PLB-985D, since the increased expression of protein was better observed when cells were pre-incubated with the proteasome inhibitor MG132. CIS and SOCS2 mRNA levels varied in cells following stimulation with the cytokines we used, but their protein expression remained stable suggesting the existence of another mechanism governing their expression. Measurement of the expression level of cytokines is important for the study of in vivo and in vitro effects of cytokines. However, sensitive and quantitative measurements of mRNA are difficult to perform because the established techniques (e.g. northern blotting) are laborious and prone to contamination. We have applied and validated a real-time quantitative PCR for cytokine mRNA quantification. This method is easy to optimize and offers sensitive, reliable and quantitative results with significantly reduced labour, allowing analysis for cytokine expression for large number of samples. We applied this method for comparing expression levels of several cytokines (TNF-a and TGF-h) and their receptors (Fas, TNFR1 and 2) between eosinophils and neutrophils isolated from peripheral blood. The results showed significance difference comparing to the volunteer samples. TWEAK is a macrophage-derived cytokine and member of the TNFa family of ligands originally identified as a weak inducer of death in certain tumor cell lines (TNF-like weak inducer of apoptosis). TWEAK exerts pleoitropic effects on a variety of cell types in vitro, including proangiogenic and proinflammatory activities. These cells express a known receptor for TWEAK, FGF-inducible molecule 14 (Fn14), which is restricted to epithelial and mesenchymal cell types, including synoviocytes and chondrocytes, and is highly upregulated in contexts of injury and inflammatory disease. It was reported that TWEAK induces human synoviocyte production of proinflammatory cytokines, chemokines and MMPs, and osteoclastogenesis of a murine macrophage cell line. Here we report that neutralizing TWEAK mAbs markedly reduce clinical paw severity in mouse and rat collagen-induced arthritis models. Inhibition by anti-TWEAK mAbs occurs at the challenge phase and does not appear to alter T cell priming or elicitation of anti-collagen antibodies. Decreased clinical paw severity correlates with reduced inflammation and protection from cartilage and bone loss at the histological level. Further studies are in progress to dissect the mechanism of action whereby TWEAK promotes joint inflammation, cartilage and bone loss and to investigate the potential interplay between TWEAK and TNF in this context. The Anti-Proliferative Effect of Infliximab on T-Cells Is Prevented by CD28 Induced Co-Stimulation. Introduction: The systemic neutralization of the proinflammatory cytokine TNFa has been shown to be beneficial for patients with RA. Since CD28+ T-cells have been implicated in the pathogenesis of RA, we evaluated if the anti-proliferative effect of Infliximab could be prevented by anti-CD28 induced co-stimulation.Aim: The aim of this study was to dissect the effect of TNFa neutralization on a well-defined cell population under highly standardized stimulatory conditions. The effect of anti-TNFa neutralization was thus examined on highly purified naRve human cord blood T-cells under different stimulatory conditions.Methods: Highly purified (N90% CD3+CD45RO-) naRve human T-cells were negatively selected from cord blood. The cells were cultured in a serum free medium (AimV) with or without anti-human TNFa chimeric monoclonal antibody (Infliximab (100Ag/mL)), with or without TGF-h1 (10ng/mL) and with or without anti-IL-10 (10Ag/mL). The cells were stimulated for four days with anti-CD3 (10 Ag/mL) with or without anti-CD28 (1Ag/mL). For analysis of cell division, T-cells were labeled with CFSE.Results: The effect of TNFa neutralization resided both on the presence of CD28 induced co-stimulation and TGF-h1. The antiproliferative effect was completely lost in the presence of anti-CD28 induced co-stimulation and strongly reduced in the presence of TGF-h1. Interestingly, the anti-proliferative potential of Infliximab exceeded that of TGF-h1 under suboptimal stimulatory conditions (without co-stimulation). Infliximab acted similarly upon both CD4+ and CD8+ T-cells whereas our earlier work has shown that TGF-h1 generally affects CD8+ T-cells more strongly than CD4+ T-cells. Finally, Anti-IL-10 did not affect proliferation except when TGF-h1 was present, independently of co-stimulation.Conclusion: The primary T-cell responses towards anti-TNFa treatment is affected by CD28 induced co-stimulation and TGF-h1. This may affect the effectiveness of Infliximab in the RA synovium where elevated levels of CD28+ T-cells and TGF-h1 have been reported.Objective: Epigenetic regulation including DNA methylation profoundly influences effector cytokine gene expression such as IFN-g and IL-4 in differentiated T helper (Th) cells. However, until now the DNA methylation status of IL-10, a cinderella immunoregulator of the immune system, remains unrevealled. Methods: A double cytokine secretion assay allowing simultaneuous isolation of cytokine-secreting cell subset of interest was used to highly purify human IL-10 + IFN-g-, IL-10 + IFN-g + , IL-10-IFN-g + and IL-10-IFN-g-human Th cell subsets after shortterm ex vivo polyclonal stimulation. Subsequently a global quantitative DNA methylation analysis of IL-10 and other Th cell differentiation related genes was perfomed. Results: Strikingly, no methylation differences were detected between these Th-cell subsets in 15,5 kb of IL-10 genomic locus spanning 9,1 kb upstream and the complete gene encompassing 99 CpGs. This included both promoter as well as other conserved noncoding regions. In contrast, hypomethylation and hypermethylation of the IFN-g gene promoter and exon I and intron I regions were noticeable in IFN-g-producers versus nonproducers, respectively. In addition, antigen-specific IL-10 + Th-cell subsets showed faded fidelity upon in vitro restimulation already after 2 weeks of in vitro expansion. Conclusion: Our results point out a lack of epigenetic memory for IL-10 expression in human Th cells. Thus, clinical applications of IL-10 + regulatory Th cells should be evaluated carefully with respect of a stable IL-10 expression in the transferred T-cell population. TNF alpha is regulated at the level of transcription, message turnover, translation and protein turnover. This has implications for chronic inflammatory diseases where the epigenetic bprintQ may be altered and contribute to disease persistence. We examined histone acetylation, histone methylation, DNA methylation and nuclear localization of the TNF alpha gene in cell lines and primary cells before and after stimulation to understand the role of epigenetics in the regulation of gene expression. Histone acetylation was not altered in our model systems by acute stimulation but was increased reproducibly by increasing maturation of monocytes. Artificially increasing histone acetylation in some settings increased TNF alpha expression but not in others suggesting histone acetylation was not sufficient for transcriptional competence. In contrast, histone K4 methylation was increased in competent cells and inhibition clearly impaired production of TNF alpha suggesting that this epigenetic mark was critical for transcription. DNA methylation correlated with competence to produce TNF alpha and was also found to be developmentally regulated and critical for production of TNF alpha. Lastly, we have used FISH to define the location of the TNF alpha locus within the nucleus. A human centromere probe was used to define heterochromatin. The TNF alpha locus was generally found in euchromatin in terminally differentiated cells, however, human stem cells which are not competent to produce TNF alpha had their TNF alpha locus in heterochromatin. From these data, we conclude there is a temporal sequence of events culminating in transcriptional competence. Early in development, the TNF alpha gene exits heterochromatin and the DNA becomes demethylated. Monocyte lineage cells further undergo modification of the H3 and H4 histones with acetylation according to their maturational state. Histone H3 lysine 4 methylation appears to be the final event leading to transcriptional competence of the TNF alpha gene. To determine whether these epigenetic marks could be altered in inflammatory disease states, we investigated patients with systemic lupus erythematosus (SLE) for evidence of altered chromatin structure. Patients with SLE had increased histone acetylation at their TNF alpha locus in monocytes compared to controls suggesting that epigenetic changes may participate in disease perpetuation. In conclusion, these data support a very strong role for TNF alpha epigenetic regulation in the control of expression of this powerful cytokine. A Fast and Robust Multiplexed Immunoassay Panel for Simultaneously Quantifying 24 Cytokines/Chemokines in Rat Serum or Plasma Using Luminex xMAP Technology. Rat is an animal model used extensively in many areas of biomedical research, where quantification of multiple cytokines and chemokines is critical for understanding physiological and pathological processes such as inflammation. However, limited sample availability, high cost, extra time and labor associated with using multiple conventional ELISA products make it impractical to measure multiple cytokines and chemokines in rat models. We previously reported a 14-plex immunoassay panel for rat cytokines, which is currently available as a commercial product. Here we report the expansion of this multiplexed immunoassay system for simultaneous quantification of 24 different rat cytokines and chemokines (Eotaxin, G-CSF, GMCSF, GRO/KC, IFNg, IL-1a, IL-1h, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-17, IL-18, IP-10, leptin, MCP-1, MIP-1a, RANTES, TNFa and VEGF) in a single serum/plasma sample of 5 AL volume. The methodology includes typical sandwich immunoassays on the surface of polystyrene beads using specific immobilized capture antibodies, biotinylated detection antibodies and streptavidin-phycoerythrin as the reporter molecule. A Luminex 100 reader is used to quantify the fluorescent signal on the beads. Each antibody pair used for individual analyte is highly specific, with no or negligible cross-reactivities to other cytokines or chemokines within the panel. The overall sensitivities are between b1 to 20 pg/mL in serum matrix. The assay robustness is demonstrated by acceptable precisions (V14% for inter-assay CV%, V8.5% for intra-assay CV%), an average recovery of 93.6 F 23% for linearity of sample dilutions, and an accuracy of 97 F 18% in serum matrix. Total assay time is 3.5 hour for serum-free samples or overnight for serum or plasma samples. The assay panel may also be used for samples such as cell culture supernatant, cell/tissue extract or other biological fluids. This simple, sensitive, accurate, and reproducible assay panel offers an economic and convenient tool for accurate and simultaneous quantification of multiple rat cytokines and chemokines in biological samples. Aging is characterized by a decline in function of multiple physiological systems. The causes of this decline are still unclear but increased oxidative stress, disturbances in energy metabolism and a primary dysregulation of the immune system might play an important role. Aging of the peripheral nervous system is associated with several morphologic and functional changes, including a decrease of the nerve conduction velocity. These changes contribute to age-related-decline in muscle strength, sensory discrimination, and autonomic responses, thereby increasing the risk of falls and disability. The aims of this study were to investigate how nerve conduction velocity in the peripheral nervous system correlates with immunological and inflammatory markers and age-associated diseases. We measured motor nerve conduction velocity of the right superficial peroneal nerve using a standard neurophysiologic technique in a population based sample of subjects aged between 20 and 103 years old taking part in the InCHIANTI study; blood count and standard biochemistry were assayed and a sample obtained for cytokines and vitamin E measurements. The InCHIANTI study examined persons living in the municipalities of Greve in Chianti and Bagno a Ripoli, two small towns located in the surroundings of Florence (Italy), randomly selected from the local population registry. Of the 1292 subjects enrolled in the study, 573 (44.3%) were males and 719 (55.7%) females. Average NCV declined with age both in men and in women. However, in each agegroup NCV was higher in women than in men. A history of diabetes (P b 0.001), stroke (P = 0.009) and peripheral arterial disease (P = 0.05) as well as the presence of cognitive impairment (0.004) were associated with a lower NCV, even after sex and age adjustment. Among the cytokines considered, no significant association with peripheral conduction could be found for Il-1h, IL-1 Ra, IL-10 and TNF-a. A significantly lower NCV was measured in subjects whose serum levels of the IL-6 (P b 0.001) as well as those of its soluble receptor (s IL-6R) (P b 0.001) were in the lower tertile. When IL-6 was adjusted for age and sex the differences among groups disappeared (P = 0.32), while for s IL-6R (P = 0.03), the adjustment changed only the strength of the association. At multivariate analysis, besides age and height, independent predictors of nerve conduction velocity included: diabetes (b0.001), cognitive impairment (0.001), sIL-6R (0.03); atocopherol (0.04), uric acid (0.01), number of lymphocytes (0.01) and neutrophils (0.004). Our results support the hypothesis that inflammation and oxidative damage are involved in the aging of the peripheral nervous system, independently from age-associated diseases. Protective Effect of Galectins Against the Generalized Shwartzman Reaction of Mice. 1 1 Cell Regulation, Kagawa University, School of Medicine, Kita-gun, Miki-cho, Kagawa, Japan; 2 Immunology and Immunopathplogy, Kagawa University, School of Medicine, Kita-gun, Miki-cho, Kita-gun, Miki-cho, Japan.Galectins are animal lectins that exhibit affinity for h-galactosides and share certain conserved sequence elements. To date, 14 galectins have been cloned in mammals and shown to play modulatory roles in diverse biological processes such as cell adhesion and proliferation, T cell apoptosis, and immune responses.Structurally, both galectin (Gal)-8 and Gal-9 belong to the tandem-repeat subfamily that is characterized by the presence of two distinct carbohydrate recognition domains (CRDs) joined by a linker peptide. It has shown that Gal-8 induces adhesion activity and superoxide production in neutrophils, and that Gal-9 possesses eosinophil chemoattractant activity as well as induces superoxide production and prolongs cell survival in eosinophils.In this study, we showed that intraperitoneal administration of recombinant Gal-8 and Gal-9 strongly induced neutrophil accumulation in peritoneal cavity of mice and Gal-8 and Gal-9 exhibited neutrophil chemoattractant activity in vitro. Neutrophil play a critical role in innate immune response and complications of bacterial infection such as septic shock and septic multiple organ dysfunction syndrome. Therefore, to determine whether galectininduced neutrophil contribute to enhance septic shock, we examined the functions of Gal-8 or Gal-9 in LPS-induced lethal shock syndrome, known as the generalized Shwartzman reaction that can be elicited by two consecutive injections of LPS. This protective effect of Gal-8 or Gal-9 against the Shwartzman reaction and suppression of these cytokine productions were diminished by depletion of neutrophils using anti-Gr-1 monoclonal antibody. Furthermore, treatment of mutant Gal-8 that lacks h-galactoside binding activity, with LPS could not suppress the elevation of IL-12, IFN-g and TNF-a levels, whereas neutrophil accumluation in peritoneal cavity of mice was observed.Thus, these results indicated that galectins modified various cytokine production induced with LPS and improved mouse mortality of the Shwartzman reaction via neutrophil accumulation. Taken together, h-galactoside binding activity of galectins was required to modify LPS induced cytokine production.Su1.20. Analysis of Cytokine Network for Initiation of Immune Responses in the Hyperplastic Thymus Associated with Myasthenia Gravis.I. 1 1 Regenerative Medicine, School of Medicine, Toho University, Sagamihara, Kanagawa, Japan; 2 Ultrastracuture, National Institute of Neuroscience, Kodaira, Tokyo, Japan; 3 Respiratoy, Japan Red Cross, Ohmori, Tokyo, Japan.Myasthenia gravis (MG) is closely associated with thymic abnormality such as hyperplasia and thymoma. Unlike experimental MG, thymectomy is effective in relieving the symptom. In particular, the hyperplastic thymus contains many IL-2R expressing B-cells, including anti-AChR antibody producing Bcells, suggesting that they are developed under the influence of hyperplastic circumstances rather than simply accumulated here from the periphery as a reservoir. In the present study we aimed to analyze thymic environment for induction of B-cell responses.MG thymi were used to examine a potential for production of Tcell cytokines regulatory for B-cell responses. For analysis of a B-cell stimulatory mechanism, nude mouse splenocytes and various cytokines detected in the MG thymus were used. In the hyperplastic thymi associated with MG, IL-2 producing cells, but not IL-4 or IFN-g producing cells, were exclusively detected, suggesting that conventional mature Th-1 cells that exclusively produce both IL-2 and IFN-g are involved at less extent in thymic IL-2 production. We found that the myoid cell conditioned medium provided a circumstance for thymic and splenic lymphocytes to produce IL-2. We also found many precursor myoid cells that produce 80-kDa and 100-kDa haemopoietic biglycan and AChR (+) cells only in their subset. These two factors and other cytokines produced by myoid cells significantly stimulated B-cell responses in a synergistic fashion with IL-2. All these results suggest that hyperplastic MG thymus contains an alternative immuno-stimulatory environment that may play an important role in breakage of anergic state against AChR. Age-Related Changes in Cytokine Production in Chernobyl Clean-up Workers from Latvia. The statement that exposure to ionizing radiation accelerates the aging process is disputable. The latest investigations confirm aging to be associated with increased inflammatory activity reflected by increased levels of circulating proinflammatory cytokines. Chronic low-grade inflammation with following impairment of immune system functions in aging promotes the development of age-related diseases, such as cancers, degenerative and infection diseases. In the same way, increased inflammatory activities have been observed after radiation exposure. Other cytokines but proinflammatory also are known to participate in the aging process. Approximately 6000 LatviaTs men were affected by ionizing radiation during they have been working in Chernobyl to clean up the after-effects of the Chernobyl power plant accident. The morbidity increases progressively among them year by year significantly exceeding that in population.Aim of the present work was to evaluate the production of several cytokines by peripheral blood cells of individuals who participated in 1986 in the clean-up work of the Chernobyl nuclear power plant explosion aftereffects depending on age.Materials and methods. ELISA measured plasma concentrations of sIL-1h and sIL-6 as well as level of IL-2 and IL-4 spontaneous and after stimulation by LPS and PHA mitogens after 24h and 96h in peripheral blood mononuclear cell culture supernatants were determined in 40 Chernobyl clean-up workers 12-17 years after their work in Chernobyl and in 40 blood-donors without a history of occupational radiation exposure. The ability of peripheral blood leukocytes (PBL) to produce IFNs was determined in 74 Chernobyl clean-up workers 12-14 years after the work in Chernobyl and in age matched 25 blood-donors. IFNs were tested in whole blood cultures by the standard virus cytopathic inhibition micromethod after their in vitro induction by Newcastle disease virus, phytohemagglutinin or doublestranded RNA. Individuals were divided in 2 age groups: the first-age 35-45 and the second-age 46-65.Results. The ability of PBL to produce IFN were significantly decreased in both Chernobyl clean-up workers age groups in comparison with blood-donors (control groups) and the incidence of inability to produce IFN in the 2 nd Chernobyl clean-up workers group two times exceeds that in the 1 st (P b 0.01). The concentration of sIL-6 was significantly higher in the 2 nd age group. The production of IL-2 as well as IL-4 by peripheral blood mononuclear cells showed no significant differences nor between both age groups or between Chernobyl clean-up workers and donors.Conclusion. The increased concentration of pro-inflammation cytokine sIL-6 together with significantly impaired anti-viral defense by decreased ability of PBL to produce IFNs in Chernobyl clean-up workers from Latvia are age dependent. Spinal cord injury (SCI) consists of two phases, instantaneous cellular destruction and axonal damage caused by the initial mechanical trauma, followed by progressive injury termed bsecondary injury,Q resulting from exposure of surrounding tissue to excitatory amino acids, cytokines, and oxidative metabolites from cellular debris or invading immune cells. It is known that increasing cyclic adenosine monophosphate (cAMP) has potent suppressive effects on the pro-inflammatory actions of the immune response, a critical component of secondary injury. Tumor necrosis factor-a (TNF-a), a central pro-inflammatory cytokine known to be activated after SCI, is important in triggering cell death and is negatively regulated by cAMP elevation. The cAMP modulation of TNF-a expression is further investigated in this study.Previous work performed in the lab has shown that the administration of rolipram, a phosphodiesterase inhibitor and a pharmacological agent capable of elevating intracellular levels of cAMP, increases the number of spared oligodendrocyte-myelinated axons after SCI. The overall goal of the current study was to elucidate putative mechanisms by which rolipram can affect TNFa initiated signaling events that mediate axo-pathology and cell death following spinal cord injury. Female Fischer rats underwent a C5 moderate contusion injury and subsequent rolipram or vehicle treatment. Animals were then sacrificed at different intervals after injury according to known TNF-a peak expression points. ELISA testing of control untreated tissue in comparison to rolipram treated demonstrated that rolipram reduces TNF-a production after SCI compared to vehicle controls. The expression of tnf-a is largely controlled through an autoregulatory loop via the transcription factor, nuclear factor kappa B (NFKB), hetero-or homo-dimeric complexes of 5 different subunits. It has been previously demonstrated that a potential shift in the balance of NFKB dimers, with an increase in p50 homodimers causes transcriptional repression culminating in reduced expression of tnf-a (Bohuslav et al., J Clin Invest. Western Blots of injured spinal cord from rolipram or vehicle treated animals were taken an hour after injury and probed for the p50 subunit of NFKB. Furthermore, in rolipram treated animals, we also found increased translocation of the catalytic protein kinase A (PKA) subunit to the nucleus. We propose that elevated levels of cAMP and downstream activation of PKA after rolipram treatment, leads to an interaction of PKA at the transcriptional control level of tnf-a that alters the balance of NFKB subunitmediated activation and repression of the tnf-a promoter. Further investigation of this interaction may lead to novel neuroprotective therapies for spinal cord injury repair.Su1.23. Analysis of IL-27 (EBI3/p28) Expression in EBV-and HTLV-1-Associated Lymphomas: Heterogeneous Expression of EBI3 Subunit by Tumoral Cells. IL-27 is a novel heterodimeric cytokine of the IL-12 family composed of two subunits, Epstein-Barr virus (EBV)-induced gene 3 (EBI3) and p28. EBI3 is expressed at high levels in EBVtransformed B cell lines, and is induced in vitro by the EBV oncogene LMP1 in an NF-kappaB dependent manner. We now show that EBI3 expression is upregulated in HTLV-1-infected cell lines and IL-2 dependent leukemic cells from Adult-T cell leukemia/lymphoma (ATL) patients, compared to normal activated T cells. EBI3 expression is decreased in HTLV-1-transformed cells by treatment with BAY11-7082, an inhibitor of NF-kappaB, and is induced in Jurkat cells by expression of HTLV-1 wild-type Tax oncoprotein, but not by a Tax mutant, M22, that is defective for NF-kappaB activation. In situ analysis of EBI3 and p28 expression in HodgkinTs lymphomas (HL), in various EBV-associated lymphoproliferative disorders (LPDs) (including post-transplant LPDs and nasal-type NK/T-cell lymphomas), and in ATL showed that EBI3 is expressed by neoplastic cells in all cases of HL and of LMP1-positive EBV-associated LPD, and at variable levels in ATL cases, but rarely in control T-cell lymphomas. In contrast, in all lymphomas tested, no or few tumoral cells expressed p28. Consistent with these data, no significant p28 and IL-27 expression was detected in HL-derived cell lines and EBV-or HTLV-1transformed cell lines. This selective overexpression of EBI3 by transformed cells suggests that EBI3 may play a role, independently from its association to p28, to regulate anti-viral or antitumoral immune responses. Inappropriate cytokine production can trigger a variety of allo-or autoimmune diseases. This is particularly true in organ transplant where immuno-suppressors like FK506 or cyclosporin A have revolutionized the field. As part of their action, these 2 drugs function as peptidyl-propyl isomerase (PPIase) inhibitors. A third member of the PPIase family, Pin1 has been implicated in cell-cycle progression but its function on cytokine expression has not been investigated. In this study, we analyzed the role of Pin1 on cytokine expression in human peripheral blood mononuclear cells (PBMC). PBMC from normal donors were activated with PHA plus PMA or anti-CD3 plus anti-CD28. Pin1 was inhibited with varying concentrations of juglone, an irreversible active site inhibitor of Pin1. At 1 AM, juglone strongly inhibited (60 to 80 % inhibition) GM-CSF, TNF-a, IL-8 and IL-4 mRNA levels by activated PBMC. At concentrations as low as 0.1 AM, juglone still reduced GM-CSF, TNF-a and IL-8 mRNAs by 65, 35 and 34 %, respectively but had little effect on IL-4. In order to further demonstrate Pin1Vs role in cytokine mRNA regulation, we transduced the WW domain of Pin1 (TAT-WWPin1) into PBMC. The WW domain affects Pin1-protein interactions and prevents Pin1 functions as a dominant negative. At 20nM, TAT-WWPin1 decreased GM-CSF mRNA accumulation by 50% but IL-4 mRNA level remained unchanged. These data suggest Pin1 is critical for the mitogen-induced elaboration of cytokines and its inhibition maybe a powerful immuno-suppressant. In tracheobronchial aspirate (TBA) of newborns, suffering from intrauterine pneumonia high levels of IL-1 h , IL-8 and TNFa (P b 0,001) were determined. The high levels of the proinflammatory cytokines accompanied by the elevation up to 23 times of neutrophil elastase (NE) and in 21 times of neutrophil myeloperoxydase (MPO). The levels of studied enzymes in control were 70 F 21 ng/ml and 250 F 75 mg/ml, respectively. Group A of patients were treated with including into traditional therapy endotracheal and intravenous infusions of recombinant IL2 (bRoncoleukinQ-Biotech, Russia). The duration of lung mechanical ventilation period of newborns of group A decreased in 2.5 times (P b 0,001), in comparison to traditionally treated patients (group B); the duration of antibacterial therapy reduced in 1.5 times (P b 0.05); hospitalization period-in 1.4 times and death rate-in 5 times. On the 7 th day of therapy a significant decrease in IL-8 and TNFa levels have been noticed in patients of group A. The concentration of IL1 h was 3 times higher than in traditionally treated group. On the 21 day of monitoring the cytokineTs and enzymeTs spectrum in TBA of rIL2 treated newborns dose not differ from healthy babies, while the levels in the control group were remain high. Cytokine and Enzyme Spectrum in Su1.26. Cytokines and Anticytokines Therapy in Severe Acute Pancreatitis.S. Chooklin, 1 A. Perejaslov. 1 1 Department of Surgery, Medical University, Lviv, Ukraine.Recent studies show an important role of cytokines in the pathophysiology of acute pancreatitis. The increased levels of proinflammatory cytokines determine progression of disease and development of its systemic complications. Imbalance between pro-and anti-inflammatory cytokines may play the pivotal role in the development of the septic complications in severe course of acute pancreatitis.The serum levels of interleukins (IL) 1-beta, 6, 8, 10, IL-1receptor antagonist (IL-1Ra), tumor necrosis factor (TNF-alpha), CD8, and CD4 lymphocytes were studied in 83 patients with acute pancreatitis.The increased levels of all pro-inflammatory cytokines at the time of admission were noted in all patients. By that, the highest levels of these cytokines were in patients with severe course of the disease. The initial levels of anti-inflammatory cytokines were higher in patients with mild pancreatitis in compared with severe once. Development of septic complications accompanies by the rapid overexpression of IL-10. Such changes in IL-10 expression in patients with septic complications may be explained by the inhibition of IL-10 the bactericidal function of neutrophils. At development of purulent-septic complications increase others antiinflammatory cytokines was observed. It was combined with is persistent imbalance of immunoregulatory T-lymphocytes with suppression predominance. Good results are received at application anticytokines therapies (pentoxifylline, dexamethasone) in initial stage of disease. In remote period there are indications to application immunoregulatory cytokines, such as interleukin 2.Thus, the therapy directed on correction of cytokines status is perspective in acute pancreatitis. Cytokine Production by Dendritic Cells Stimulated with Microbial Products Modulated by Epigallocatechin Gallate (EGCG). J. Rogers, 1 I. Perkins, 1 A. van Olphen, 1 N. Burdash, 1 T. W. Klein, 1 H. Friedman. 1 1 Medical Microbiology and Immunology, University of South Florida College of Medicine, Tampa, FL, USA.Dendritic cells (DCs) are important monocytic phagocytes essential for innate immunity, especially to opportunistic intracellular microorganisms like Legionella pneumophila. In this study DCs were stimulated in vitro by either infection with Legionella or the microbial stimulant LPS from E. coli or MDP from Gram positive bacteria. Production of the Th1 helper cell activating cytokine IL-12 and the proinflammatory cytokine TNFa was rapidly induced. These cytokines are important for antimicrobial immunity and were assessed in the DC cultures by ELISA assay. Treatment of the stimulated DCs with EGCG, the primary polyphenol catechin in plant products, inhibited in a dose dependent manner production of IL-12 important for activating immune cells against intracellular bacteria like Legionella. In contrast, treatment of the DCs with EGCG enhanced production of TNFa in a dose dependent manner. TNFa is important in inflammation and considered crucial for the inflammatory response. Thus the results of this study show that EGCG, the major catechin widely present in plant products such as green tea, has marked effects on production of immunoregulatory cytokines by stimulated DCs known to be involved in antimicrobial immunity, especially innate immunity. Further studies are warranted to determine the mechanisms of the effects of this and other catechins on cytokine production by immune cells, such as DCs. TSG-6 protein, the secreted product of TNF-stimulated gene 6 (TSG-6), is an important endogenous mediator of inflammation and female fertility. Previous studies have shown that TSG-6 has a protective effect in murine models of experimentally induced arthritis, but the mechanism responsible for this effect has remained elusive. To gain insights into the role of TSG-6 we investigated whether TSG-6 protein affects the expression of inducible cyclooxygenase-2 (COX-2), a key enzyme in inflammation and immune responses. We found that TSG-6 protein upregulates the COX-2 protein expression in the RAW 264.7 murine macrophage cell line. The effect of TSG-6 was abolished by heat denaturation, trypsin digestion, or anti-TSG-6 specific antibodies. TSG-6 treatment of cells also resulted in a rapid increase in COX-2 mRNA levels, suggesting that TSG-6 up-regulates COX-2 gene expression. The PGD 2 metabolite, 15-deoxy-D 12,14 -PGJ 2 , can act as negative regulator of inflammation through the activation of the nuclear peroxisome proliferator activator receptor-g, and inhibition of multiple steps in the nuclear factor-nB signaling pathway. Thus, induction of COX-2 expression in macrophages and preferential PGD 2 synthesis may underlie the protective effect of TSG-6 observed in experimental models of arthritis. Hyaluronan, a molecule with which TSG-6 interacts, modulates the COX-2inducing activity of TSG-6 protein. This work provides the first demonstration that TSG-6 can influence the expression of a gene regulating inflammation in a cell culture system, which may help to gain a better understanding of the physiological and pathophysiological functions of TSG-6. Results: The 4-1BBL gene had been introduced successfully into mouse gastric carcinoma cell lines MFCs. The oncogenicity of MFC/4-1BBL cells was obviously lower than that of MFC/ pMKITneo cells and wild-type MFC cells. The peripheral blood NK cells and CD4 + T cells in mice inoculated with MFC/4-1BBL cells were notably more than that in mice inoculated with MFC/ pMKITneo cells and MFC cells, but to CD8 + T cells had no difference as compared with before injection of MFC/4-1BBL cells.Conclusions: The oncogenicity of MFC/4-1BBL cell distinctly declines, and it can induce the proliferation of NK cells and CD4 + T cells in tumor-bearing mice. This study provides experimental basis for the further developing of the gene-modified tumor vaccine. Objectives: To evaluate the effects of mIL-23 gene modified murine colon carcinoma cell (Colon26/IL-23) vaccine on macrophages and T cells.Materials and Methods: Colon26/IL-23, Colon26/ LXSN(transfected with vector) and Colon26 cells were treated with mitomycin C to prepare tumor cell vaccines. BALB/c mice were immunized with those vaccines by i.p. Griess assay was used to detect the level of NO in peritoneal macrophages. The proliferation of macrophages and splenic cells was determined using ([ 3 H]-TdR) incorporation method. Macrophage mediated tumor cytotoxicity and CTL activity were assessed by radioactive ([ 3 H]-TdR) release assay and LDH assay respectively. immunized with the vaccines were also observed.Results: Cytotoxicity of macrophage and CTL activity to tumor were enhanced in the mice i.p. immunized with Colon26/IL-23 vaccine and the level of NO secreted by the macrophages from the mice injected with Colon26/IL-23 vaccine was higher than those in controls. The Colon26/IL-23 vaccine also promoted the proliferation of macrophages and splenic cells and increased survival time of mice which were challenged with wild Colon26 cells.Conclusions: mIL-23 gene modified tumor vaccine can activized macrophages and T cells and also has anti-tumor effect. Objectives: To examine whether expression of mIL-23 in murine colon carcinoma cells (Colon26/IL-23) could induce murine macrophage to secrete NO and TNF-a.Materials and Methods: Normal BALB/c mice were injected by i.p. with Colon26/IL-23, Colon26/LXSN (transfected with vectors), and Colon26 cells respectively. On days 5 and 10 after the cell implantation, peritoneal macrophages were prepared by lavage with HBSS and collecting the adherent cells. NMA (N Gmonomethyl-L-arginine) and LPS were inhibiter and inducer of NO production respectively. The production of NO was determined by Griess assay. The levels of TNF-a and IL-12 were detected by ELISA. Some mice were used for observation of survival.Results: The levels of NO and TNF-a produced by the macrophages from the mice injected Colon26/IL-23 cells were higher than those from control mice at both time-points. The production of NO and TNF-a of the LPS stimulated macrophages from the mice injected Colon26/IL-23 cells was more than those in control groups. The level of IL-12 of the macrophages from the mice injected Colon26/IL-23 cells is similar to controls. The average survival time of the mice injected Colon26/IL-23 cells is longer than that of control groups. Regulation of Inducible Heparanase Gene Expression in Human T-Cells by Soluble and Immobilized TNFA. Ilya Sotnikov, Liora Cahalon, Oded Vainas, Rinat Eshel, Ben-Zion Katz, Neta Ilan, Israel Vlodavsky, Irun Cohen, Ofer Lider. 1 Immunology, The Weizmann Institute of Science, Rehovot, Israel; 2 Bone Marrow Transplantation, The Hematology Institute, Sourasky Medical Center, Tel-Aviv, Israel; 3 Oncology, Hadassah-Hebrew University Hospital, Jerusalem, Israel.In the course of an inflammatory response immune cells need to acquire directional migrating phenotype. This is done by means of degrading extracellular mesh with enzymes, and by promoting adhesion and migration of the cells by active molecules, such as cytokines. One of the enzymes is heparanase, a heparan sulfate specific endoglycosidase, secreted by immune cells, which combines cytokinelike properties with conventional enzymatic activity. Upon acidification of cellular environment heparanase degrades sugar chains, whereas at basic pH it increases adhesion of the cells to extracellular matrix and promotes their movement along chemokine gradients.Given the versatile functions of this molecule, finding the prerequisites for its gene expression regulation is important. Our major goal was to investigate inducible heparanase gene transcription in primary human T-cells in response to exogenous signals.In our work we utilized 95% pure CD3+ T-cells, derived from healthy human donors. As an activator we used TNFa, a major proinflammatory cytokine, which delivers a stress signal to cells, bearing TNF receptors, and influences their transcriptional programs. T-cells were exposed to soluble TNFa, or the cytokine was first pre-complexed with fibronectin, and then the cells were placed on this immobilized TNFa.We measured the heparanase mRNA levels and protein content in cell lysates using real-time quantitative PCR, and heparanase enzymatic assay, respectively.The cytokine in its soluble and fibronectin-immobilized form promotes the accumulation of heparanase mRNA and protein in a strictly defined time-and concentration-dependent manner. To assess the intracellular signaling pathways, involved in TNFa-induced signal transduction, necessary for the heparanase gene transcription, we used chemical components, known to inhibit key players of signal transduction cascades, initiated by TNF receptor ligation. We show, that the heparanase mRNA accumulation depends on PKC, p38, JNK, PI3k signaling cascades, whereas it is not influenced by NFkB and ERK. To further strengthen the notion of specificity of TNFa signaling, we also demonstrate, for the first time, a strong correlation between the effects of active TNFa concentrations on the upregulation of relevant transcription factors, such as GATA-3, Ets-1, Egr-1, but not Ets-2, measured by immunoblotting, and heparanase mRNA accumulation. Finally, we show, that these effects are reproduced, although to a limited degree, by other members of TNF superfamily, FasL and TRAIL, suggesting the involvement of intracellular signaling components, shared by these ligands.In conclusion, we claim that heparanase, an important agent in the autoregulatory loops of inflammatory lesion, is responsive to the signals, delivered by the common stress signals, such as TNFa. Serum has been essential for both freezing and testing of human PBMC. Serum contains a plethora of bioactive molecules whose variable concentrations makes every serum batch unique, with unique affects on T cell performance in vitro. We developed a serum free protocol for freezing PBMC. The thawed PBMC displayed N 80% viability regardless whether they were frozen with serum, or serum free. Moreover, the thawed PBMC maintained full functionality compared to the fresh cells when tested in ELISPOT assays against a panel of 23 individual peptides and 5 protein recall antigens recognized by CD8 and CD4 cells, respectively. Strikingly, the PBMC performed frequently better under serum free conditions: increased numbers of cytokine producing cells were elicited by the recall antigens without an increase of activity in the medium control. The magnitude of signal enhancement under serum free conditions was considerably larger than with inclusion of costimulatory antibodies, such as anti-CD28. Apparently, serum contains suppressive factors (such as IL-10 and TGF-beta) that can interfere with T cell activation. Thus, serum free freezing and testing media are not only a convenient alternative to bypass the need for screening for boptimialQ serum batches, but also such media enhance and standardize T cell monitoring. Hepatitis C virus (HCV) infection is the leading cause of chronic liver disease in the U.S., affecting approximately 4 million people. The impact in healthcare-related costs is enormous, with over $600 million being spent annually in loss of work and medical costs. The immune mechanisms that lead to chronic liver damage and cirrhosis in hepatitis C have not been elucidated but are increasingly being attributed to nonapoptotic, unscheduled cell death and release of a variety of DAMPs including uric acid, S100 proteins, heat shock proteins, and HMGB1, each acting in a cytokine like role with specialized immunoglobulin type receptors including RAGE and TLR2 and TLR4. HMGB1 is a highly conserved nuclear protein that has a surprising extracellular role. It binds DNA, increasing access to transcription factors, and also recruits cells across endothelial barriers, promoting local production of TNF, IL-6, and IFNg. It is released from necrotic cells, activated macrophages, NK cells and mature DCs, but not neutrophils. Following DNA damage as a result of apoptotic death, ultraviolet irradiation, or platination, it is sequestered in the nucleus. Detectable in serum and tissue it serves as a leaderless cytokine, prototypic of the DAMPs that drive repair and a previously underappreciated role in inflammation. HMGB1 was measured in a highly sensitive sandwich ELISA developed in our laboratory and did not correlate with other serum analytes including ALT, AST, total bilirubin, or albumin. Pretransplant HMGB1 was detectable in the serum of all 17 patients evaluated, ranging from 9.9-487.1 ng/ml including three pts with hepatoma (62.6, 74.1, and 485.9). We evaluated whether HMGB1 correlated with disease activity at baseline or at 3, 6, 9 or 12 months of follow-up with the presence of hepatoma, histologic activity index [HAI], rejection activity index or stage (degree of fibrosis). Following orthotopic liver transplantation, HMGB1 levels diminished at 3mo. One pt with a baseline level of 74.1ng/ml with hepatoma increased post transplant to 581.3ng/ml and is being reevaluated for recurrence. We conclude that HMGB1 represents an independent measure of tissue damage and injury, as well as cancer that may be useful in the assessment of patients with HCV infection. Controlling HMGB1 activity and release is an approach being developed as an experimental therapy for patients with sepsis, arthritis, cancer, and other inflammatory disorders. Ex Vivo Pro-Inflammatory Cytokines Expression in Patients with Aggressive Periodontitis. 1 RATIONALE: Periodontitis includes a broad spectrum of immuno-inflammatory responses to periodontal pathogens from oral biofilm that destroy the supporting tissue of the teeth. In this pathology the balance between pro and anti-inflammation is tipped to pro-inflammatory activity mediated by cytokines that include TNFa and IL-1h. OBJECTIVE: We explored the relationship of TNFa and IL-1h expression and the aggressive periodontitis (AP) condition in Chilean patients. For cytokine expression blood culture were stimulated with LPS from E. coli or P. gingivalis extracts. TNFa and IL-1h were measured by an ultrasensitive ELISA kit.RESULTS: The LPS-induced TNFa levels in AP patients were higher than controls (P = 0.0414) while means of serum TNFa concentrations in patient group were 3 times higher than those found in controls (P = 0.000). Although no significant the highest mean for LPS-induced IL-1h level was obtained by the AP group.CONCLUSION: The mononuclear cells from AP patients express higher induced TNFa and IL-1h levels than healthy individuals in an ex vivo culture system. Interleukin(IL)-22 was discovered in 2000. It belongs to the IL-10 family of cytokines whose members (additionally IL-10, IL-19, IL-20, IL-24, and IL-26) are structurally related molecules. We have previously shown that IL-22 is mainly produced by activated T cells, particularly the Th1 subset. The data presented here surprisingly show that IL-22, unlike IL-10, does not act on immune cells. This conclusion is based on a systematic study at all possible levels of analysis: on receptor expression, signal transduction, effects in vitro, and effects in vivo. In contrast, the quantitative analyses of a wide range of tissues and corresponding primary cells and cell lines showed that many non-immune tissue cells are target of IL-22 as they express both chains of the IL-22 receptor complex. Very high levels of IL-22 receptor chains were found in skin and keratinocytes. In primary human keratinocytes these levels were further upregulated by IFN-g suggesting an increased sensitivity of these cells towards the IL-22 action under T1 conditions. The receptor complex on these cells was functional and induced STAT3 tyrosine phosphorylation. For the first time, this study also identified effects of IL-22 on keratinocytes, namely the upregulation of the antimicrobial agents b-defensin 2 and b-defensin 3. This effect was transcriptionally regulated, and independent on protein de novo synthesis and alternative protein secretion indicating a direct effect of IL-22. Additionally, this induction was time-and dose-dependent, and enhanced upon cellular differentiation. The extent of induction was comparable to that by other known inducers of b-defensins. In skin from patients with psoriasis, high levels of IL-22 were highly significantly associated with strongly upregulated expression of b-defensin-2 and b-defensin-3 suggesting a protective effect of IL-22 in this disorder. Taken together, IL-22 does not serve the communication between immune cells but is a T cell mediator that directly promotes the innate, non-specific immunity of the skin.The observation that activated T1 cells directly regulate the non-specific immune defense in tissues demonstrates a so far unknown but very important side of the immune system. Human B cell production has been thought to differ from that in mouse with respect to the requirement for IL7. In mice, IL7 increases the in vitro production of B cells from adult murine bone marrow by 20-30 fold. In contrast, human studies have focused on B lymphopoiesis from fetal precursors and show little increase in B cell production with IL7 (~2 fold). A re-examination of IL7 knockout mice has shown that IL7 is required to generate adultderived B2 cells (CD5-), but not for the production of fetallyderived CD5+ B1 cells. Here we examine the role of IL7 in human B cell development from hematopoietic stem cells (HSCs) isolated from human umbilical cord blood (CB), a source likely to contain both HSCs that give rise to B1 cells, as well as those capable of generating B2 cells. FACs analysis of human CB shows mutually exclusive expression of IL7Ra and CD5 on CD34+CD19+ pro-B cells. When FACS-sorted Lin-CD34+ CB HSCs were placed in stromal cell co-cultures, the addition of IL7 increased the in vitro production of human B lineage cells up to 60-fold as compared to cultures with anti-IL7 neutralizing antibody. IL7-induced increases observed on human stroma were~10-fold more than those with the murine S17, MS-5, or OP9 stromal cell lines. A titration of IL7 into co-cultures showed that the threshold for IL7-induced increases in human B cell production was 10 to 100 fold lower in co-cultures with human stroma as compared to those with murine stroma. Surprisingly, murine stromal cells provided better support for human B cell production in the absence of murine or human IL7 activity. Our data show that IL7, in the presence of human stroma, can dramatically impact B cell production from at least a subset of progenitors in human CB. On the other hand, murine stroma support IL7-independent B cell production from human CB progenitors. Using Ki-67 and Annex V/7AAD staining we are examining the extent to which proliferation and/or anti-apoptotic effects, at the pro-B and pre-B stages, contribute to IL7 effects seen in vitro. Our studies suggest that the roles of IL7 in human and murine B cell development may be more similar than previously believed. Objectives: A functional polymorphism in the chemokine receptor CX 3 CR1 has recently been associated with protection from coronary artery disease (CAD) and internal carotid artery (ICA) occlusive disease in man. CX 3 CR1 is functionally expressed on monocytes and vascular smooth muscle cells (VSMC), both of which cell types are critical for the vascular remodeling found in atherosclerosis, angioplastic restenosis, and ICA occlusive disease. Our objectives in this study were to investigate whether CX 3 CR1 was involved in the inflammatory process of vascular remodeling, and the mechanisms by which it may play a role. Methods: Femoral arteries of CX 3 CR1-deficient and wild type mice were subjected to injury by the passage of an angioplasty guidewire or sham operation. After 5, 14, and 28 days, mice were euthanized, perfused with 4% paraformaldehyde, and tissues harvested for immunohistochemistry. In some cases, mice were injected with bromodeoxyuridine (BrdU) for 24 hours prior to tissue harvest for cell proliferation studies. At d5, the injured arteries of WT mice had a marked monocyte infiltration into the intima compared to non-injured arteries on the control side of each animal (13.6 F 11.5 vs. 0.0 F 0.0, P = 0.006). At d14, WT injured arteries had neointimal hyperplasia with a mixture of monocytes and VSMC. By d28, the intimal hyperplasia comprised primarily of VSMC with an average intima to media ratio (I/M) of 0.78 F 1.04 compared to 0.0 in non-injured arteries (P = 0.02). In CX 3 CR1 -/mice, there was no detectable monocyte invasion to the intima at d5 (100% decrease compared to WT, P = 0.006), and there was an 86.8% decrease in the numbers of monocytes in the intima at d14. At d28, the intima area was decreased in CX 3 CR1 -/mice by 62% compared to WT mice (P = 0.11), with a concomitant decrease in the numbers of VSMC in the intima (87.1%, P = 0.055). As expected, the number of VSMC in the media declined during the injury response in WT animals (33.4% decline, P = 0.025) while it remained unchanged in the media of CX 3 CR1 -/mice (9.9% decline, P = 0.22). The percentage of actively proliferating cells in injured vessels was also decreased in CX 3 CR1 -/mice (12% vs. 27% in WT, P = 0.027). Conclusions: CX 3 CR1 plays a critical role in vascular remodeling following femoral artery injury. Although the VSMC defects seen at d28 were downstream of the monocyte defects observed at d5, we can not rule out a possible contribution of CX 3 CR1 to the proliferation and media-tointima migration of VSMC in the course of vascular remodeling. It is generally assumed that most mammalian genes are transcribed from both alleles in most situations. But it is now clear that several genes do not have this transcriptional behavior. In general these genes are grouped in 3 different classes, a) imprinted genes, b) most genes in the X chromosome of female cells and c) autosomal genes that are stochastically monoallelic transcribed. Interleukin 10 (IL-10) is a multifunctional cytokine, its activities array from the classical inhibition of effector functions of T lymphocytes, monocytes and macrophages, to regulation of growth and differentiation of B lymphocytes and dendritic cells, and its recent key role in differentiation and function of the T regulatory cell. Making use of IL-10eYFP ki mice, in which transcription from different alleles is easily distinguishable, we observed that IL-10 shows a pseudo-monoallelic transcription pattern in CD4 + T cells. Most CD4 + T cells express stochastically only from one allele while only few transcribe it from both. TCR signaling through activation of transcription factors and regulation of chromatin opening seem to act together to provide efficient control of IL-10 expression. We think that this two-layer regulation might reveal an extremely important mechanism in controlling genetic expression in biological situations of low frequency expression of genes and as such might apply to many more genes than the few ones studied so far. Interleukin-1 (IL-1) is one of the main mediators involved in the control of inflammation and acute phase response. IL-1 family molecules regulate tissue repair, inflammatory and immune reactions. Important question of IL-1 biology today is how to use this pleiotropic highly biologically active molecule in the therapy of various human diseases. Systemic IL-1 administration induces adverse effects limiting IL-1 therapeutic use. Very promising is IL-1 local application just to the lesion or inflammatory site. Human recombinant IL-1 beta has been used for local therapy in patients with chronic bacterial purulent rhinosinusitis after failure of routine antibiotic therapy. Endogenous IL-1 beta and IL-1 receptor antagonist (IL-1RA) ex vivo production and gene polymorphism studies for the distribution of IL-1b and IL-1RA alleles were performed in all patients. Patients with more rare IL-1b (+3953) + allele had elevated IL-1 beta production and patients with IL-1RA 2*(VNTR) + allele had higher antagonist production compared with patients bearing normal alleles. These abnormal alleles were found in 30% and 90% of patients respectively that was much more frequently compared to healthy subjects suggesting for genetic predisposition to chronic rhinosinusitis probably due to the disregulation in the IL-1 family cytokines production. We have found that recombinant IL-1 therapy was highly effective in patients carrying IL-1RA 2*(VNTR) + allele with increased endogenous IL-1RA levels. In these patients IL-1 beta local application significantly increased functional activity of leukocytes isolated from the inflammatory sites tested in the assays of neutrophil migration to fMLP, superoxide production, adhesion and phagocytosis. Cytological analysis showed that the reduction in inflammatory manifestations was accompanied with the decrease in the proportion of neutrophils and increase in the numbers of monocytes/macrophages. Negative results of IL-1 beta therapy was obtained in patient who had very rare combination of homozygous IL-1b (+3953) + and normal IL-1RA 2*(VNTR)-allelic variant that led to elevated IL-1 beta production and normal IL-1RA levels. According to obtained results IL-1 family cytokines gene polymorphisms are linked to chronic rhinosinusitis and may influence results of cytokine immunotherapy. Background: Hypersensitivity pneumonitis (HP) is a complex lung syndrome of varying intensity and clinical presentation that result of an immunologically-induced inflammation in response to a large variety of inhaled antigens. HP is characterized by a remarkable T-cell alveolitis. However, the evaluation of lymphocyte phenotypes, primarily CD4 + /CD8 + T cell subsets has given inconsistent results. Likewise, studies of other T-cell phenotypes in human HP like T-helper 1 (Th1) versus Th2 are scanty. We hypothesized that some divergence in T-cell subsets may play a role. We analyzed a variety of T-lymphocyte phenotypes in cells obtained by bronchoalveolar lavage of subacute and chronic patients.Methods: Forty one patients with HP induced by avian antigens and classified as subacute (21 patients), or chronic (20 patients), 5 healthy subjects.HP BAL cells were stained with mAbs to CD3, CD4, CD8, CXCR3, CCR4 for surface staining and intracellular IFN-g, IL-2, IL-4, IL-10. The antigen specific secretion of IFN-g, IL-2, IL-4, IL-10 following pigeon serum stimulation was determined by CBA, and this was used to identify specific CD4 Th1 and Th2 subpopulations. In order to precise the best cut point of CD4 + /CD8 + ratio, an analysis of their sensitivity versus 1specificity (ROC curves) was used. The test with higher sensitivity and the lower 1-specificity was considered to be the most useful.Results: BAL CD4 + /CD8 + ratio was highly heterogeneous, and although higher levels were revealed in patients exhibiting chronic HP, results did not reach statistical significance (3.5 F 3.7 versus 1.7 F 1.5 and 1.2 F 0.4 from subacute patients and controls respectively). However, 14 chronic patients showed values over 2.0 compared with 7 subacute patients. A significant difference in the receptor chemokine expression was noticed. Thus, subacute patients exhibited significantly higher numbers of T-cells expressing CXCR3 (40.6 F 10.7 % versus 25.6 F 7.3% in chronic cases). By contrast, CCR4 was higher expressed in chronic patients 6.0 F 1.3% versus subacute 2.1 F 1.3%.Intracellular IFN-g/IL-4 ratio was significantly higher in subacute patients 1.6 F 0.9% versus chronic 0.7 F 0.45% after antigen specific stimulation. Also IFN-g /IL-10 ratio measured in supernatant cultures was significantly higher in subacute patients 152 F 24.2 versus chronic 0.6 F 0.7. Evaluation of T H 1/T H 2 Cytokine Modulations in Chronically HIV-Infected Adults Who Received Therapeutic Vaccination and Intermittent HAART Following an Initial HAART Intensification (CTN-140 Pilot Trial). Sardar Sindhu, 1, 2, 3 Maude Loignon, 1 Evaluation of T H 1/T H 2 cytokine modulations is important to determine the immune competence and/or restoration in chronic HIV patients undergoing initial highly active antiretroviral therapy (HAART) intensification (GM-CSF, Hydroxyurea, and ddI), therapeutic vaccination (Remune TM ) and structured treatment interruption (STI). We evaluated serum levels of interferon (IFN)-gamma, interleukin (IL)-2, tumor necrosis factor (TNF)alpha, IL-15, IL-4, and IL-10 in 10 patients enrolled in the Canadian HIV Trials-140 pilot study. All patients were in viremic control, i.e., virus load (VL) was b 50 copies mL À1 at the time of baseline samplings. Nine sampling points were at baseline, HAART-intensification, therapeutic vaccination, 1 st peak VL (rebound during STI), 1 st b 50 VL, 2 nd peak VL, 2 nd b 50 VL, 3 rd peak VL, and 3 rd b 50 VL, respectively. All cytokines were measured by competitive enzyme-linked immunosorbent assays (ELISA). The difference between group means at a given timepoint and at baseline was determined by t-test. We found that IFNgamma levels were comparable to baseline value of 58.88 pg mL À1 at all time points except at 2 nd peak VL and at 2 nd b 50 VL samplings when the levels were significantly reduced. IL-2 levels were elevated significantly (P b 0.05) at 1 st time as VL was below 50 copies mL À1 compared with baseline value of 58.43 pg mL À1 and high levels (higher than in age-matched HIV-seronegative controls) were sustained thereafter. Conversely, IL-10 levels were reduced significantly at 1 st time as VL was b 50 copies mL À1 than baseline concentration of 109.90 pg mL À1 and low levels were maintained at all subsequent time points. Regarding both TNFalpha and IL-4, all modulations were found to be statistically nonsignificant (p N 0.05) as compared with baseline mean concentrations of 124.0 pg mL À1 and 75.35 pg mL, À1 respectively. However, IL-15 levels were comparable to baseline concentration of 241.10 pg mL À1 (which was higher than those in controls) until 1 st time as VL was b 50 copies mL À1 and the levels were significantly reduced thereafter. Therefore, it was concluded that these immunomodulatory therapeutic interventions were able to induce/maintain a T H 1-dominant cytokine profile in these patients whereas suppressed IL-15 levels at 2 nd peak VL and thereafter might be due to a deregulated innate immune response. The Monocyte Locomotion Inhibitory Factor (MLIF), could so contribute to the sparse delayed inflammation observed in amebic abscess of the liver, either directly through its effects upon MP (i.e depression of chemotaxis and respiratory burst), or indirectly through the modulation of the production and/or secretion of cytokines involved in the recruitment of MP in the late stages of inflammation. We evaluated the effect of MLIF on the production of pro/anti-inflammatory cytokines in CD4+ cells.Materials and Methods T CD4+ lymphocytes were purified by negative selection using a commercial kit (MACS-ReagenTs isolation of human CD4+ T cells. One  10 7 PBMC cells were placed in propylene tubes with 80ml PBS-albumin-EDTA and 20 Al antibody cocktail to eliminate cells other than CD4+ (10 min at 4 8C). The CD4+ lymphocyte purified fraction was found to be 95% pure. MLIF (96% pure) was commercially obtained (American Peptide Co, Sunnyvale, CA, USA). Five  10 5 T CD4+ cells were placed in RPMI-1640 with 10% fetal calf serum (FCS) and were stimulated for 24 h in the presence of PMA (50 ng/ml), MLIF(50 Ag/ml) or PMA+MLIF. The supernatant fluids were analyzed by ELISA.Results Pro-inflammatory cytokines. Cells activated with MLIF expressed an increased production of constitutive pro-inflammatory cytokines IL-1h, IL-2 and IFN-g (262, 245, 46 pg/ml respectively) when compared with the production of constitutive basal RPMI cell controls (26, 18, 10 pg/ml respectively). PMA enhanced the induced production of the same pro-inflammatory cytokines (192, 384, 84 pg/ml respectively) . The expression of IL-1h and IFN-g induced by PMA was significantly inhibited by MLIF (25% and 20% respectively), which did not occur with IL-2.Anti-inflammatory cytokines. MLIF, increases the production of constitutive anti-inflammatory cytokines when compared to basal IL-5, IL-6 and IL-10 (188, 114, 134 pg/ml respectively) when compared to the basal production in control cells RPMI (15, 16 and 13 pg/ml respectively). With PMA, an increase of production of induced IL-5, IL-6 and IL-10 (575, 250, 326 pg/ ml respectively), while the mixture of PMA+MLIF was found to decrease the production of constitutive PMA of IL-5 and IL-6, but not of IL-10, that significantly increased its expression.Conclusions Pro/anti-inflammatory cytokines were produced by MLIF, in amounts similar to those induced by PMA. With the PMA+MLIF mixture the IL-1h, IFN-g, IL5 and IL-6 (all with pro-inflammatory potential) production was inhibited, but not that of IL-10 (the prototype of an anti-inflammatory cytokine), which disclosed a significantly increase of its expression. Thus MLIF can orchestrate an anti-inflammation pattern of cytokines that contribute to the exiguous inflammation found in advanced lesions of invasive amebiasis (CONACYT GRANT 38104-M). Interleukin 21 (IL-21) is secreted by activated T cells and modulates immune cell functions with both pro-and antiinflammatory effects. Interleukin 21 receptor, (IL-21R) homologous to IL-2R beta and IL-4R alpha, associates with gamma common chain upon ligand binding. IL-21R is constitutively expressed on many cell types in the immune system, and is upregulated by IL-21. Rationale: Since blockade of the IL-21 pathway with soluble IL-21RFc resulted in a reduction of clinical signs of arthritis in rodent models, we examined the effect of this pathway on rat and murine responses in vitro to understand the potential mechanisms of IL-21 regulation in arthritis. Animals were treated with soluble mIL-21RFc, which neutralizes both murine and rat IL-21 bioactivity. Draining lymph node cells from collagen-immunized mice were cultured in vitro with collagen and either IL-21 or IL-21RFc and assayed for proliferation and cytokine secretion. Cytokines and anti-collagen specific IgG levels were also measured in the serum by ELISA analysis. In the rat, Con A stimulated spleen cells or anti-CD3 activated splenic T cells were assayed for proliferation and cytokine secretion in response to IL-21 and IL-21RFc. Results: When compared with control animals, 3x weekly treatment with IL-21RFc resulted in less severe signs of disease in both rat and murine prophylactic and therapeutic models of CIA. Addition of murine IL-21 to collagen restimulated LN cells resulted in a dose dependent inhibition of proliferation, and a decrease in IFN-gamma, GMCSF, and IL-6 and increase in IL-10 in the culture supernatant. Conversely, addition of IL-21RFc enhanced the collagen proliferation response and resulted in increased IFNgamma, GMCSF and IL-6 and decreased IL-10 production suggesting that IL-21RFc modulated endogenous IL-21, as well other antigen-induced cytokines. Conclusion: These results suggest that Interleukin 21 plays a role in modulation of antigen-specific T cell responses and this may contribute to the pathology of arthritis. Objective: To investigate the molecular mechanisms of neural stem cells (NSCs) responses to the proinflammatory cytokine interferon-g.Background: Self-renewal capacity is a fundamental property of NSCs, in which stem cells proliferate forming multipotent neurospheres in the presence of FGF-2. NSCs respond to inflammation, however it is not known what molecules account for their response during disease. Our hypothesis is that NSCs express functional immune related genes that interact with stemness genes, and control NSC intrinsic properties.Materials and Methods: We analyzed multiple microarray datasets for inflammatory genes expressed by NSCs then performed gene clustering by Gene Ontology, and data mining using several bioinformatics databases. For testing self-renewal capacity, NSCs were isolated from different regions of mouse brain, and cultured in DMEM/F12 with N2 supplement and bFGF (20 ng/ml). For gene expression analysis mRNA from duplicate NSC cultures exposed to interferon-g for 24 h, were processed on Affimetrix microarrays. Genes showing a change in expression of N2-fold with a p-value of P b 0.001, were confirmed using a different microarray platform, confocal microscopy and western blot.Results: We found that NSCs express several interferon related genes, we confirmed their expression in vivo on E14.5 germinal zone by confocal microscopy. IFN-g at doses of 50 to 500 U/ml added to NSC cultures from E14.5, newborn, or adult brain, decreases the frequency of FGF-2 induced neurosphere formation (39.6 F 9.2% vs. 4.6 F 3.5%, P b 0.002) and neurosphere diameter (131 F 33.2 vs. 42.2 F 14, P b 0.0001).To determine the genes affected by IFN-g, primary NSCs were treated with IFN-g and subjected to microarrays. We identify N100 affected genes, clustered in 3 groups by a Terrain Gene Map: a) IFN-g highly inducible genes (absent in untreated NSCs with 20-2000 fold increase) b) MHC genes and proteosome genes increased 2-10 fold c) Decreased genes, 50% of which are related to stemness including cell cycle, histone and lifespan genes. We confirmed the expression of selected genes by confocal microscopy and western blot. Interferon-g induced rapid phosphorylation of STAT1 Tyr-701 and nuclear translocation of STAT 1 in NSCs 15 min after treatment. Instead, stat1-/-brains contain more NSCs and IFN-g in vitro increased their neurosphere formation by 3-fold (self-renewal capacity). Experiments of differential gene expression and behavior in vivo of stat1-/-vs wildtype mice after IFN-g treatment are under way to further evaluate the importance of stat1 in NSCs.Conclusion: NSCs have a functional interferon genetic program. Interferon-g induces this program and leads to the activation of multiple genes including stat-1, which appear to form a central checkpoint for NSCs self-renewal capacity.Su1.49. Role of CXCL1/CXCR2 in Promotion of Glial Survival during Experimental Demyelination.L. T. Remington, 1 S. P. Zehntner, 1 C. A. Hollmann, 1 T. Owens. Inadequate repair is thought to underlie chronic demyelination in Multiple Sclerosis (MS). Oligodendrocyte precursor cells (OPCs), which are present in the adult central nervous system (CNS), have the ability to migrate into lesions and differentiate into mature oligodendrocytes. OPC proliferation and differentiation is driven by growth factors such as the chemokine CXCL1. We have examined the role of CXCL1 and its receptor (CXCR2) in the corpus callosum of female C57BL/6 mice during cuprizone-induced demyelination. Response to the demyelination causes astrocytes, microglia and OPCs to accumulate in the corpus callosum. We found a transient increase in CXCL1 mRNA in the forebrain prior to OPC accumulation, as early as 1 week after the initiation of cuprizone treatment. The level of CXCL1 mRNA peaked at two weeks and then declined to normal levels. CXCR2 positive cells also increased in the corpus callosum during demyelination. PDGF receptor alpha positive OPCs were shown to express CXCR2, suggesting a possible mechanism for migration of these cells into the corpus callosum. To test this, we injected a replication-defective adenovirus encoding CXCL1 via the cisterna magna which introduced the virus into the cerebrospinal fluid without inflammation. Virus was injected at 0 weeks or 3 weeks after initiation of cuprizone treatment and mice were sacrificed at either 3 weeks or 6 weeks. Virally encoded CXCL1 mRNA was detected in the cerebellum up to 6 weeks after virus injection. Immunohistochemical detection of NG2 positive OPCs showed no effect of the CXCL1 encoding adenovirus on the number of OPCs in the corpus callosum. Deletion strategies are currently underway. Standardizing Multiparameter Flow Cytometry for Evaluation of Cytokine-Secreting Activity in T Cells Via Automation of Sample Preparation and Analysis. 1 1 Biomedical Research Division, Beckman Coulter Inc., Miami, FL, USA. The utility of evaluating T cell functional assays is well recognized in the context of determining an efficacious vaccine strategy for infectious diseases/cancer, a tolerance profile in autoimmunity and transplantation, as well as for understanding the basic mechanisms of T cell immune responses in disease pathogenesis. However, the variability associated with T cell functional assays continues to be problematic, especially in studies involving multi-center clinical trials as well as in longitudinal studies. The variable natures of these assays are associated with a variety of factors ranging from source of the T cells, the processing methodology (isolation, freezing, thawing, and culturing), the sample preparation for staining and finally, data analysis, and data reduction. With a view to reducing variability and standardizing targeted steps of the assays involving T cell function, an automated methodology for staining and analysis of intracellular cytokines via flow cytometry was developed.A modification to available sample preparation instruments was performed that enabled the automated pipetting, incubation, and staining of intracellular and surface molecules of human stimulated whole blood or PBMC for flow cytometric analysis. A 5-color flow cytometry assay (2-3 surface markers; 2-3 intracellular cytokines) was developed to characterize the brestricted polyclonalQ Th1 versus Th2 cytokine profile response in T cells stimulated by SEB/CD28. The complex nature of the cytokine profile and inter-and intrasubject variability was revealed on evaluation of several cytokines in the context of multiparametric evaluation of the T cell responses. The use of automation thus provides a greater degree of standardization in these functional assays, allowing for a correlation of variability and complex immune response profiles, to vaccine efficacy or disease progression without as significant an interference due to the variable nature of the manual assay. High endothelial venules (HEVs) are specialized lymph node blood vessels that allow entrance of lymphocytes into lymph nodes. Through our analysis of genetically deficient and transgenic mice, we have previously determined that the LTah complex is critical for expression of several HEV genes during development and that the LThR is expressed on HEV. The specific objectives of this study were to determine: 1) whether LThR signaling is crucial for HEV gene expression in adult wild type mice and 2) whether this is a direct effect on endothelial cells in induction of HEV gene expression. These questions were investigated in vivo in C57BL/6 mouse peripheral lymph node (PLN) and in vitro in the bEnd.3 cell line. The regulation of HEV genes (GlyCAM-1, MAdCAM-1, SLC, an HEV specific GlcNAc-6-sulfotransferase , and LThR itself) was evaluated by immunohistochemistry and real time PCR, using an LThR activator, an LThR inhibitor and contact sensitization. HEV genes were regulated by contact sensitization with oxazolone in C57BL/6 mice. LThR was regulated in parallel with HEC-6ST after contact sensitization with oxazolone. An interesting kinetic pattern was detected for these and several other HEV genes: there was an initial decrease in gene expression and a rebound by 7 days in most of the genes with the exception of MAdCAM-1, where there was an exactly opposite effect. The initial phase of gene regulation after contact sensitization was independent of TNFRI, though the optimal rebound of gene expression was partially dependent on that receptor. LThR signaling was essential for the optimal rebound of gene expression after sensitization. bEnd.3, a mouse endothelial cell line, prominently expressed the LThR and upon stimulation with a hamster anti-LThR antibody showed a dramatic up-regulation of MAdCAM-1. These in vitro and in vivo data taken together indicate that the LThR is expressed on endothelial cells, can signal direcly, is crucial for constitutive HEV gene expression and is regulated during an immune response. These data also provide information regarding plasticity of HEV gene expression during an immune response, suggesting an initial reversion to an immature phenotype and recovery through a developmental program to an adult phenotype.Supported by NIH CA 16885 to NHR and the Anna Fuller Fund (SL).Su1.52. Interferon-A Inducing TLR9 Agonists. Bacterial and synthetic DNA containing CpG dinucleotides (CpG DNA) have been shown to be the natural ligands for TLR9, a molecular pattern recognition receptor. CpG DNA activation of TLR9 has been shown to produce Th1 type immune responses, including IFN-a induction. Through extensive DNA structure-activity relationship studies, we have identified novel synthetic dinucleotides and DNA structures (immunomers) that are recognized by TLR9. In general, IMOs produce distinct TLR9-mediated immune responses with high IL-12 and low IL-6 cytokine secretion profiles compared with natural CpG dinucleotide motif. Based on human cell culture assays and in vivo non-human primate studies, we have identified novel IMOs consisting of synthetic stimulatory motifs, which induce high levels of IFN-a. The induction of IFN-a by IMOs is dependent on the presence of specific nucleotide sequences and the activation of plasmacytoid dendritic cells. These novel IMOs also induced human B cell proliferation and activated B cells to express surface markers and secrete cytokines. IMOs induced expression of a number of cytokine and chemokine genes, including IFN-a gene and a number of interferon inducible genes such as 2V5V-adenylate synthetase in human PBMCs. In non-human primates, IMOs administered s.c. at 1 mg/kg dose induced high levels of IFN-a secretion up to 72 hr. These results suggest that IMOs that induce high levels of IFN-a may be suitable candidates for the treatment of hepatitis C and cancer, where recombinant IFN-a therapy is commonly used. Although CD4+CD25+ regulatory T cells (Treg) are instrumental in the maintenance of immunological tolerance, one critical question is whether CD4+CD25+ regulatory T cells (Treg) can only be generated in the thymus or can differentiate from peripheral CD4+CD25-naRve T cells. We have shown that conversion of naRve peripheral CD4+CD25-T cells into CD4+CD25+ regulatory cells can be achieved through co-stimulation with TCR and TGF-beta. These converted regulatory cells are not only unresponsive to TCR stimulation, but also inhibit normal T cell proliferation in vitro. Importantly, TGF-beta-converted transgenic CD4+CD25+ suppressor cells proliferate in response to immunization, but inhibit antigen-specific naRve CD4+ T cell expansion in vivo in an OVA peptide TCR transgenic adoptive transfer model. In a murine asthma model, co-administration of TGF-beta-induced suppressor T cells prevents house dust mite (HDM)-induced allergic pathogenesis in lungs. Significantly, TGF-beta induces Foxp3 gene expression in TCR-challenged CD4+CD25-T cells, which mediates their transition toward a regulatory T cell phenotype. The TGF-beta induction of Foxp3 is attributed to the CD4+CD25-naRve T cells, but not selective expansion of a small number of pre-existing regulatory T cells. Accordingly, TGF-beta induced-Foxp3 expression in naRve CD4+CD25-T cells can be detected as early as 16 hours after stimulation. TGF-beta induces Foxp3 expression in CD4+CD25-T cells in the 5C.C7 TCR transgenic mouse on a Rag2-/-and IL-2-/-background. TGF-beta 1-/-mice exhibit reduced number of peripheral CD4+CD25+CD62L+ T cells. Finally, CD28 signaling is involved in TGF-beta induction of Foxp3 in CD4+CD25-T cells. The data provide new evidence in understanding the mechanisms for generation and development of CD4+CD25+ regulatory T cells, and open a unique opportunity to intentionally manipulate this pivotal population of cells for immune tolerance and T cell-based immunotherapy. Osteoprotegerin Synthesis by Circulating T Cells Is Related to Osteoporosis in HIV-Infected Patients. 1 Osteoprotegerin (OPG), a cytokine of the TNFR superfamily, is the decoy receptor for Receptor Activator of NF-nB ligand (RANKL). OPG counteracts bone resorption induced by interaction of the receptor-ligand pair RANK-RANKL. OPG, present in blood, is also implicated in diverse aspects of vascular function and in immune regulation. The aims of this study are to better understand the different functions of circulating OPG through an elucidation of the role of immune cells in its regulation and to explore new aspects of interactions between the immune and skeletal systems. Our objectives, formulated in this broad framework, would be:1. To delineate the subpopulation(s) of peripheral blood mononuclear cells responsible for the synthesis of osteoprotegerin and to identify the factors of regulation.2. To study the functional impact of OPG regulation by immune cells in an appropriate physiopathological context i.e in HIV infected individuals.To address these issues we used enzyme immunometric assays combined with immunoblots to examine the synthesis and the regulation of OPG by subpopulations of normal human peripheral blood mononuclear leucocytes (PBMCs), enriched by immunomagnetic separation, under different conditions of incubation. RNA synthesis profiles were studied using RT-PCR and Northern blot analyses. We provide evidence that, amongst PBMCs, OPG is constitutively secreted only by CD4+ T lymphocytes, and that T cell production of OPG is up-regulated by an anti-CD3 antibody, IL-1h, TNF-a, GM-CSF, vitamin D 3 , but not TGF-h. The T cell synthesis of OPG is strongly increased by IL-4, unlike IL-10 which inhibits the constitutive and IL-4-induced production of OPG. To verify the pathophysiological importance of T cell-derived OPG, we chose to study circulating concentrations of OPG of patients infected with HIV-1 knowing that disease induced by HIV-1 is specifically associated with both compromised CD4+T cell number & function, and an increased incidence of osteoporosis. Given that OPG is presently considered one of the final components that decides outcome of bone resorption, we compared circulating concentrations of OPG in HIV-infected individuals to agematched normal subjects and remarked a significant decrease of OPG concentrations in plasma from HIV-infected patients. Additionally, normal T lymphocytes in the presence of recombinant HIV-1 gp120 had an unequivocal reduction of their constitutive OPG production. Moreover, nelfinavir, a protease inhibitor used in highly active antiretroviral therapy, significantly inhibited the constitutive OPG synthesis by normal T cells. These results are the first to suggest a causal link between HIV-induced T cell malfunction, i.e a dysregulation of its capacity to produce OPG, and secondary osteoporosis. We highlight a new role for T lymphocytes in the regulation of circulating OPG, and suggest the therapeutic potential of OPG or OPG mimetics in the decreased bone mineral density associated with HIV infections. Detection of Cytokines in Patients with Hypersensitivity to Metals.Z. 1 1 Clinical Department, The Institute of Dental Research, 1st Medical Faculty and GUH, Charles University, Prague 2, Prague, Czech Republic. Introduction: In the dental praxis, dentist are often confronted with suspect delayed hypersensitivity reaction to various metals used in prosthetic and restorative dentistry. Cytokines have been involved in most disease processes including hypersensitivity. In this study a new method of detection of cytokines so called bHuman Cytokine ArrayQ was used in ten patients with suspect delayed hypersensitivity to metal components of dental alloys.Methods: The clinical part of study involves examination of the oral cavity with respect to the presence of dental alloys including dental radiograph. Special questionaire focused in general health status was also used. In the immunological examination mononuclear cells separated from peripheral blood were used to study the production of cytokines. By using Ray Bio TM Human Inflammation Antibody Array III it was possible to identify the expression profiles of 40 cytokines. The detection of cytokines was performed after 3 days cultivation with mercury and nickel chlorides.Results: Nickel chloride stimulated production of IL-6, IL-10, IFN-gamma, IL-1 alfa, TNF alfa and TNF beta. Mercury chloride stimulated production IL-4, IL-6, IL-10, IL-1 alfa and sTNF RII.Conclusion: Mercury chloride activated rather clones of TH2 lymphocytes, while nickel chloride TH1 lymphocytes. Human CCL4/MIP-1beta and CCL3/MIP-1alpha are two highly related molecules that belong to a cluster of inflammatory CC chemokines located in chromosome 17. CCL4 and CCL3 were formed by duplication of a common ancestral gene, generating the SCYA4 and SCYA3 genes, which, in turn, present a variable number of additional non-allelic copies (SCYA4L and SCYA3L1). Moreover, our group found that SCYA4L locus is polymorphic and displays a second allelic variant (SCYA4L2) highly represented in HIV+ patients compared to the one described originally (SCYA4L1).Recent evidences have pointed out that variation in SCYA3L1 gene copy number play a key role in susceptibility to HIV infection. Probably SCYA4L will be also related with this phenomenon. Similarly this gene dosage variation could be involved in other diseases where these chemokines play important roles.We developed a FRET probes Real-time PCR based method to determine easily, fast and reliably the genotype for the polymorphic SCYA4L locus. This polymorphism consists in a single nucleotide change situated in the second intron acceptor splice site (+590ANG). Our approach uses specific primers for SCYA4L locus and a pair of FRET hybridization probes. We have tested the use of the sensor probe complementary to each one of the allelic variants, obtaining a better melting resolution with the SCYA4L2 specific probe, while SCYA4L1 sensor probe is more useful for total SCYA4L copy number determination. Based on this approach we are setting up a protocol to determine the copy number of the SCYA4L gene whereas it informs about copy number belonging to SCYA4L1 or to SCYA4L2 allelic variants. Correlation of Interleukin-4 and Chronic Periodontitis. 1 1 Immunology, Shaheed Beheshti University Medical School, Tehran, Islamic Republic of Iran.Aim: Regarding to the presence of numerous B cells in chronic periodontitis and since TH2 cells have important role in inducing the humoral responses, so the aim of this study was to determine the correlation between IL-4 (as a most important cytokine for differentiation of TH2 cells from TH0 cells and IL-12 (as a most important cytokine for differentiation of TH1 cells from TH0 cells) and chronic periodontitis.Materials and Methods: For this purpose, 20 gingival samples from healthy patients and 20 gingival samples from patients with chronic periodontitis were collected during periodontal surgery. Tissue samples were cultured for 72 hours, then ELISA was used for determining the concentration of IL-4 and IL-12 in supernatant fluids.Results: IL-4 and IL-12 were found in all of samples. There was no significant difference between case and control groups regarding IL-4 or IL-12 concentration. But there was significant difference between two groups regarding the ratio of IL-4 to . Also there was correlation between IL-4/IL-12 and attachment loss (P~0.028).Conclusion: It is concluded that in chronic periodontitis, probably the number of TH2 cells is more than TH1 cells but in active phases of inflammation and tissue damage, the number of TH2 cells could be decreased by the reduction in IL-4 production.Su1.58. Interleukin-21 Maintains T Cells in a Naive Phenotype.S. Ferrari-Lacraz, 1 D. C. Foster, 2 R. Chicheportiche. 1 1 Immunology and Allergy, University Hospital, Geneva, Geneva, Switzerland; 2 Cytokine Biology, Zymogenetics, Seattle, WA, USA.Of the large number of cytokines released during the development of the immune response, T cell growth factors (TCGFs) such as interleukin-2 (IL-2), IL-15 and IL-21 play major and distinct parts in T-cell activation. These TCGFs determine whether activated T cells undergo apoptosis or persist as regulatory/memory cells after robust immune activation, the key to peripheral T-cell homeostasis.IL-21 is a major TCGF and part of the innate and adaptive immune response. IL-21 co-stimulates naive, but not memory T cells, contrary to IL-15 that is a major stimulus of memory CD4+ and particularly CD8+ T cells. Unlike IL-2 and IL-15, IL-21 has no significant effect on the proliferation of T cells in the absence of anti-CD3 or other stimuli. Here we demonstrate that IL-21 plays a potent role in maintaining the naive phenotype of human CD4+ T lymphocytes, with the persistence of specific cell-surface markers. PBMC were isolated from healthy young donors, CD45RO-(naive) and CD45RO+ (memory) CD4+ T cells were subsequently isolated on a FACSvantageW sorter. The selected CD4+ cells were cultured for up to 3 weeks with IL-2 or IL-21. The phenotype of naive and memory CD4+ T cells was assessed by immunofluorescence. Markers such as CD45RA, CD27, CD62L and particularly CCR7 were clearly upregulated in CD4+ T cells cultured with IL-21 as compared to IL-2. Less than 1% of CD4+ T cells cultured with IL-2, but 43% of CD4+ T cells cultured with IL-21 express CD45RA at day 21. This means that the majority of naive CD4+ T cells cultured with IL-2 switched to a CD45RO+ memory cell phenotype by day 21. The expression of CCR7 on CD4+ T lymphocytes cultured with IL-21 induces the migration of these cells through the chemoattraction of CCL21. Moreover, IL-21 reduces the frequency of proliferating naive T cells compared to IL-2, but proliferation markers, such as Ki-67 (B56) and proliferating cell nuclear antigen (PCNA) were expressed equally in both cell-culture conditions. Therefore, IL-21 is a unique TCGF able to maintain CD4+ T lymphocytes under a naive phenotype, and to increase their cellsurface expression of CCR7 and their migration through chemoattraction to CCL21. These findings suggest that IL-21 may play a unique role in innate and adaptive immune response due to the persistence of naRve T lymphocytes. Osteopontin (OPN), previously regarded as an adhesive bone matrix protein, has recently been shown to be a pleiotropic cytokine regulating early cell-mediated immunity. Our previous studies have shown OPN to play a critical role in autoimmune demyelinating diseases. In multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) OPN expression is significantly increased in the affected tissues. Here we show that OPN inhibits TCR-mediated apoptosis in vitro without affecting cellcycle progression as measured by BrdU incorporation and CFSE labeling. To investigate the effect of OPN on activation-induced T cell death in vivo, we activated Vh8(+) T cells by injecting Staphylococcal enterotoxin B (SEB) into OPN -/mice and monitored the deletion of Vh8(+) T cells. We found that the death of these stimulated T cells in vivo is accelerated in OPN -/mice. Adoptive transfer of T cells from OPN -/mice to Rag-1 -/recipients revealed that OPN secreted from T cells prolongs survival of activated T cells in vivo. We also found in OPN -/mice that expression of the pro-apoptotic Bcl-2 family protein, Bim, was increased in both CD4 + and CD8 + T cells. Furthermore, compared to wild type mice, OPN -/mice display a remarkably reduced number of certain T cell subsets. In OPN -/mice, in comparison to the T cell subpopulations CD62L(lo)CD44(hi) and CD62L(lo)C-D44(int), CD62L(hi)CD44(hi) express higher levels of prosurvival protein Bcl-2. Despite of increased cell death in OPN -/-, after T cell activation the level of active caspase-3 was lower in OPN -/cells than wild type cells. Furthermore, treatment of OPN -/and wild type lymphocytes with a cell-permeable caspase inhibitor reduced the death of activated wild type but not OPN -/-T cells, which suggests that activation-induced cell death in OPN -/-T cells is mediated by an alternative, caspase-independent pathway. In this study, we suggest that OPN plays a role in regulating programmed cell death of activated T cells and that OPN signaling promotes T cell survival. Comparison of RNA Preparation Methods and Their Effect on Cytokine/Chemokine Gene Expression. A. L. Asare, 1 S. A. Kolchinsky, 1 P. Wood, 2 V. L. Seyfert-Margolis. 1 1 Immune Tolerance Network, University of California, San Francisco, San Francisco, CA, USA; 2 Center for Human Genetics and Integrated Biology, University of Pittsburgh, Pittsburgh, PA, USA.Objective: Commercially available blood collection tubes for gene expression studies lyse red blood cells and stabilize RNA to ensure detected transcript profiles reflect the physiological state of the subject. We assessed two such systems for their ability to detect and quantify differential expression of cytokine and chemokine genes. Method: Peripheral blood samples from 8 healthy volunteers were collected in both Tempus Tubek blood collection tubes (Applied Biosystems (ABI), Foster City, CA) and PAXgenekBlood RNA Tubes (PreAnalytiX, Valencia, CA). All samples were isolated according to the manufacturersT instructions. RNA purity and yield were assessed prior to analysis. Samples were preprocessed as follows: 1) Blood was drawn directly into Tempus k and PAXgenek tubes or 2) Samples were drawn in LI Heparin tubes, stimulated with PHA, and then transferred into the two RNA collection tube types. We analyzed both: 1) Transcript detection at single time-points and 2) Differential expression between baseline samples and samples stimulated with 25 Ag/ml PHA after 3 hrs. Affymetrix HG-U133 2.0 GeneChipn with 47K transcripts and quantitative RT-PCR for 184 cytokine and chemokine transcripts (probe primers from ABI) were run. Results: Initial analysis of 2 out of 8 participants generated a GeneChipn analysis set of 1,245 reliably detected transcripts. Clustering using standard correlation generated the following differential expression profiles: Set A (370 transcripts) showing up regulation using both tubes; Set B (336 transcripts) showing down regulation using both tubes; Set C (180 transcripts) showing no change, but having elevated baseline values in Tempusk but not PAXgenek; Set D (249 transcripts) showing no change, but having elevated baseline values in PAXgenek but not in Tempusk. Transcripts in Set C and D were not cytokine/ chemokine related. Transcripts in Set A with greater than 10 log scale expression include chemokine (c motif) ligand 1, class 1 MHC-restricted T cell associated molecule, chemokine (C-X-C motif). Quantifying differential expression by RT-PCR showed high similarity between the tubes for all cytokine/chemokine transcripts. Some allergy relevant cytokines, such as IL-5, were not detectable at high levels in the GeneChipn assay, but were detected at high levels using RT-PCR. Conclusion: Different kinetics for each tube during the lysing process may account for detection level differences at single time-points for non-cytokine/ chemokine related transcripts found using GeneChipsn. These time-point differences could not be confirmed using RT-PCR since cytokine/chemokine RT-PCR probes were used. This demonstrates that the selection of a particular tube for RNA studies may depend heavily upon the transcripts of interest. For cytokine/chemokine transcripts, both tube types provide essentially equivalent expression profiles. Cytokine networks play a crucial role in the disease progression of both multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). The study of these networks is necessary for understanding the disease and for developing treatments. However, cytokines are multifaceted and may fulfill contradictory roles depending upon a variety of factors, e.g. Tumor necrosis factor (TNF) is an important cytokine in both EAE and MS. Mice that lack TNF receptor type 1 (TNFR1 KO) develop mild EAE but express high levels of interferon gamma (IFN-gamma) in the spinal cord. IFN-gamma is a pro-inflammatory cytokine traditionally considered detrimental in MS, but which has been shown to be protective in EAE. Wild type (WT) or TNFR1 KO mice were immunized with MOG35-55 peptide in complete FreundTs adjuvant. Seven to ten days later, lymph node T cells were isolated by MACS using negative selection. Splenic antigen presenting cells (APCs) from unimmunized mice were also obtained by MACS using negative selection. WT or TNFR1 KO T cells were co-cultured with WT or TNFR1 KO APCs in the presence of MOG35-55 peptide, and cytokine expression was analyzed at various time points by ELISA or real-time PCR. Both WT and TNFR1 KO T cells produced significantly more IFNgamma when co-cultured with TNFR1 KO APCs compared with WT APCs. This observation was not altered by the addition of neutralizing anti-TNF antibody, demonstrating that the enhanced IFN-gamma secretion was not due to TNF actions through TNFR2. Furthermore, these data demonstrated that TNFR1 signaling on the APC is critical in regulating IFN-gamma production. APCs can regulate T cell IFN-gamma production by secretion of IFN-gamma-inducing factors, such as IL-12. Preliminary results show that mRNA expression of the p40 subunit of IL-12 was higher in co-cultures containing TNFR1 KO APCs compared to WT APCs. This suggests that the absence of TNFR1 on APCs leads to higher levels of IL-12, which results in higher IFN-gamma secretion by T cells. IL-12 promotes IFN-gamma-secreting T cells, whereas IL-23 promotes IL-17-secreting T cells that have recently been shown to be critical for EAE pathology. Protein array analysis revealed high levels of IL-17 in our co-cultures, and future work will further investigate the role of IL-17 in our coculture system. In conclusion, this study shows that lack of TNFR1 on APCs leads to greater IFN-gamma production by T cells, potentially due to a change in p40 expression. These data further extend our understanding of the cytokine networks underlying CNS inflammation. This study was funded by the Canadian Institutes of Health Research and the Multiple Sclerosis Society of Canada. General Autoimmunity Su1.62. Bacterial HSP70 Immunization Inhibits Proteoglycan-Induced Arthritis.S. E. Berlo, 1 C. B. ten Brink, 1 P. J. van Kooten, 1 R. van der Zee, 1 M. Singh, 2 B. J. Prakken, 3 T. T. Glant, 4 W. van Eden, 1 C. P. Broeren. 1 Immune responses to heat-shock protein 70 (HSP70) are seen in inflammatory diseases, such as rheumatoid arthritis (RA), diabetes and atherosclerosis. Although HSPs have well described roles as chaperones for intracellular proteins, the significance of immune responses to HSP is only now becoming clear and indicate that HSPs are targets for disease suppressive immune regulation. In experimental disease models HSP can be used as therapeutic agents to prevent or arrest inflammatory damage and first clinical trials using HSP peptides have shown a shift in pro-inflammatory cytokine profiles of peptide specific T cells to production of antiinflammatory cytokines. In order to analyze the mechanisms leading to HSP induced immune regulation we set out to test the possible protective effects of HSP70 in a novel mouse (BALB/c) arthritis model. injection of 100 Ag mycobacterial HSP70 given 10 days prior to the first PG injection (both given in DDA) resulted in a two-week delay of the arthritis onset. Furthermore, HSP70 preimmunization induced a significantly milder arthritis, as assessed by clinical score (mean maximum arthritis score 0.7 F SEM 0.4; compared with 3.5 F SEM 1.3 in the control mice; P b 0.001). The protective effect of HSP70 was accompanied with HSP70-specific T cell proliferation, IFN-g and IL-10 production, in mice pretreated with HSP70. Interestingly, in these mice we could also detect IL-10 production against the arthritis inducing proteoglycan, which was not present in control mice. In addition, joint sections of HSP70-pretreated mice showed less leukocyte infiltration, reactive synovial cell proliferation and cartilage destruction compared to the control group.In conclusion, HSP70-initiated modulation, could dramatically suppress the development of inflammation and subsequent tissue damage in PGIA. In addition, we demonstrated that HSP70 preimmunization, indeed, activated T cells, altered the cytokine profile and modulated the immune regulatory response. Characterization of Functionally Important Regions of CD200:CD200R for Immunoregulation Using Blocking Synthetic Peptides and/or mAbs. Objective: We have previously defined the immunoregulation which occurs, allowing prolongation of allograft survival and suppressing collagen-induced arthritis in mice, following interaction of the cell surface molecules, CD200 and CD200R. The CD200R1 isoform is most important in delivering direct immunosuppressive signals. Other groups have used a biophysical approach to investigate the structurally important regions for these interactions, and have suggested the N-terminal (extracellular domains) of the molecules are crucial. We report on our investigations of the important interactions using an approach investigating purturbation of various biological functions which follow CD200:CD200R interactions.Materials and Methods: A series of 15-mer peptides were synthesized which defined continuous sequences of the extracellular region of the murine and human CD200 molecule. In addition, peptides mapping to the presumptive CDR1, CDR2 and CDR3 of the human and mouse CD200R1 molecules were synthesized. We assessed the ability of these different molecules to block the interaction of CD200 with CD200R1 in a competitive ELISA using plate-bound CD200R1Fc and biotinylated CD200Fc, as well as by FACS using FITC-conjugated CD200Fc binding to 24hr-LPS-activated adherent cells. Peptides were also used to modulate the suppression of MLC reactivity in vitro which follows from inclusion of CD200Fc in MLC cultures, measuring development of CTL after 5 days of culture.In addition, using a newly prepared panel of mAbs to mouse and human CD200Fc, we compared the rank activities of antibodies for binding (FACS or ELISA) to CD200 with their abilities to augment immune reactivity in MLCs.Results: Peptides defining discrete regions in the N-terminal regions of CD200 and CD200R1 were functionally active in the different assays, including the assay which investigated blockade of CD200Fc-mediated suppression. Moreover, infused in vivo, the same mouse-specific peptides suppressed protection from graft rejection afforded by injection of soluble immunosuppressive CD200Fc.Using the panel of mAbs, we observed again that only mAbs defining epitopes in the N-terminal domain could augment MLC reactivity (or block immunosuppression by soluble CD200Fc), while mAbs targeting C-domain epitopes, although reactive in ELISA or FACS (targeting cell surface CD200) were inactive in MLCs. In a final assay we compared by rank these different anti-CD200s for FACS staining of CD200 expressed on various cell types, including fresh dendritic cells and lymphoid tumor cells. These data suggested there exists a significant heterogeneity in expressed CD200.Summary: Our data confirm that the N-terminal regions of CD200 and CD200R1 are crucial to mediate immunoregulation, and suggest that it will prove possible to generate smaller molecular weight antagonists of this interaction which may have clinical utility. Introduction: Anti-phospholipid syndrome (APS) is an autoimmune disease characterized by thrombotic and hemocytopenic manifestations in patients with high titers of autoantibodies directed against phospholipids and phospholipids cofactor proteins. Molecular studies of anti-phospholipids (aPL) had been performed on monoclonal autoantibodies obtained from EBV transformed B cells, which do not represent the repertoire of the autoimmune aPLs. Using phage display technology is possible the study of a wide repertoire of V H (D)J H and V L J L rearrangements that recognize specific antigens. Objective: To study by phage display the V H (D)J H and V L J L rearrangements of anti-g 2 GP-I and anti-Pt from a patient with PAPS with high titers of these autoantibodies.Methods: cDNA was synthesized from RNA obtained from peripheral blood mononuclear cells. The V H DJ H and V L J L (n and E) rearrangements were amplified. Heavy and light chains fragments were linked to obtain scFv fragments. The scFv were ligated to a pSyn2 vector. E. coli XL1-Blue were transformed with the products of the ligation. Cultures were co-infected with the M13/KO7 phage to rescue the antibodies in the format phage-scFv antibody. Four rounds of selection were done against g 2 GP-I and Pt. Positive clones were sequenced and analyzed.Results: Two clones that recognize g 2 GP-I (beta 1 and 2) and one that recognize Pt (Prot-1) were obtained. The number of somatic mutations suggests that the rearrangements are the product of antigen driven clones. A Novel Network of Human B Cell Effector Cytokines Is Implicated in Autoimmunity: Dysregulation in Patients with Multiple Sclerosis and Potential as Therapeutic Target.A. Bar-Or, 1 M. Niino, 1 M. Duddy, 1 F. Adatia, 1 S. Hebert, 1 H. J. Kim. 1 1 Neuroimmunology Unit, Montreal Neurological Institute, McGill University, Montreal, QC, Canada. Background: Beyond their roles as potential antibody producing cells, there is growing interest in the fundamental roles that B cells may play in regulating immune responses. Emerging animal studies point to an important contribution of B cell effector cytokines to autoimmunity. We set out to study the potential involvement of human B cells and their cytokines in immune regulation and autoimmunity.Results: We report that the profile of human B cell cytokine production is context dependent, being critically influenced by the balance of signals through the B cell receptor and CD40. B cells appropriately stimulated by sequential B cell receptor and CD40 stimulation proliferate and secrete tumour necrosis factor (TNF)-a and lymphotoxin (LT) which contribute to germinal center reaction and thereby amplify the ongoing immune response. In contrast, CD40 stimulation alone-a mimic of a B cell receiving bystander T cell help in the absence of specific antigen recognition-induces significantly less pro-inflammatory cytokines (n = 24; P b 0.0002) but significantly enhanced production of IL-10 (P b 0.004) that serves to suppress inappropriate immune responses. We further observed that this novel network of reciprocal regulation of B cell effector cytokines, is abnormal in a subgroup of patients with relapsing remitting multiple sclerosis where B cells maintain their capacity to proliferate and produce the proinflammatory cytokines TNF-a and LT (n = 18; P = 0.46 compared to normals), but have a significantly diminished capacity to produce IL-10 (n = 18; P b 0.02). This B cell cytokine network can be targeted therapeutically, as evidenced in patients treated with mitoxantrone (NovantroneR), a recently approved immune suppressant in MS. Compared to B cells from untreated patients, B cells from mitoxantrone treated MS patients produce significantly less of the pro-inflammatory cytokines TNF-a (n = 8; P b 0.02) and LT (P b 0.05), but significantly more of the anti-inflammatory cytokine IL-10 (P b 0.05). We further observed that treatment was associated with a decrease in the proportion of circulating memory B cells, suggesting that the apparent reciprocal regulation of B cell cytokines mediated by mitoxantrone, related to differential effects on distinct B cell subsets. This guided further studies of the fundamental B cell cytokine network and enabled us to ascribe unique effector cytokine profiles to normal memory and naive human B cell subsets.Summary: We ascribe active roles for memory and naive human B cell subsets in appropriately promoting or suppressing local immune responses in the normal state through production of distinct effector cytokines. We further suggest that dysregulation of this B cell cytokine network occurs in some patients with the human autoimmune disease multiple sclerosis, and that this network represents a potential therapeutic target. Activation of autoreactive B cells is pivotal for the initiation and propagation of many autoimmune diseases. It is generally thought that this activation reflects a breakdown in peripheral tolerance, although the nature of the development of these cells is not completely understood. Many studies in B lymphopoiesis have shown that IgM receptors drive development and construction of naive B cell repertoire, whereas IgG receptors promote formation of the memory B cell compartment. This isotypic change results from a gene rearrangement process called class switch recombination (CSR), and is thought to occur in mature peripheral B cells upon activation and providing of appropriate T cell help. In the absence of T cell help, activated B cells undergo Fas-mediated apoptosis, a process thought as a peripheral mechanism for selftolerance. We have recently shown that CSR spontaneously occur during normal B lymphopoiesis in vivo and in vitro, and that these cells acquire a memory phenotype. We found that CSR in B lymphopoiesis is T cell-independent, but further expansion of the cells is regulated by T cells. To further study these cells we have used the Igm-deficient mouse model (mMT), where B cell development is limited by the ability of the cells to undergo CSR. We found that isotype switched B cell precursors can mature to the periphery and differentiate to antibody-producing plasma cells. These B cells generate a gH-driven repertoire that is oligomonoclonal and bearing self-tissue reactivity. Further studies revealed that isotype-switched B cell precursors are negatively selected by Fas signaling, as blocking the Fas/FasL interaction rescues the development of isotype-switched B cells in vivo and in vitro. Our studies propose a novel developmental pathway driven by gH-isotypic receptor that effectively circumvents peripheral tolerance requirements. This pathway, however, is strictly controlled by the Fas signaling to prevent B cell autoimmunity.Su1.67. The Role of VCAM-1 in Self-Reactive T Cell Fate. Vascular Cell Adhesion Molecule 1 (VCAM-1) plays a role in leukocyte migration and adhesion, and is found on a variety of cells including endothelial cells, thymic epithelium, and dendritic cells. VCAM-1 binds to its principal ligand, Very Late Antigen-4 (VLA-4) found on B cells and T cells. Disruption of VCAM-1/VLA-4 signaling is associated with prolonged allograft acceptance and impaired T cell responses. Therefore a role has been established for VCAM-1 as a positive factor for T cell activation and proliferation. It is our hypothesis that in the absence of VCAM-1 expression on dendritic cells, antigen specific, self-reactive CD4 + T cells will fail to proliferate as effectively as when VCAM-1 is present. It is also hypothesized that some self-reactive T cells activated in the absence of VCAM-1 will survive and acquire a regulatory T cell phenotype. To test this hypothesis we will use 3A9 antigen specific T cells that recognize Hen Egg Lysozyme (HEL) protein presented as peptide by antigen presenting cells. Transgenic mice expressing membrane-bound or soluble HEL have been crossed onto conditional VCAM-1 knockout mice lacking functional expression of VCAM-1 on all subsets of splenic dendritic cells. By adoptive transfer of labeled 3A9 T cells into either HEL or VCAM -/-HEL recipient mice, the fate of these antigen specific T cells can be followed over time. Most modified lipoproteins circulate as antigenantibody complexes (immune complexes, IC) that can be characterized after precipitation with 4% PEG. Chemical and immunological analysis of IgG and LDL isolated from IC demonstrated that the human immune system recognizes malondialdehyde lysine, carboxymethyl lysine and other yet uncharacterized epitopes. The IgG fraction isolated from precipitated IC contains antibodies reactive with copper-oxidized LDL (oxLDL) and AGE-modified LDL (AGE-LDL) predominantly of the pro-inflammatory subclasses 1 and 3. Comparison of the IgG subclass and dissociation constants of antibodies contained in precipitated IC and remaining in the supernatant after PEG precipitation showed that antibodies included in IC have statistically significant higher avidity and relatively higher IgG1 concentrations. Affinity chromatography isolation of circulating antibodies to oxLDL and AGE LDL showed that IgG antibodies predominated over IgM antibodies, and that IgG subclasses 1 and 3 predominated over subclasses 2 and 4, for both antigens. Because IgG1 and IgG3 antibodies can activate complement and deliver activating signals to phagocytic cells as a consequence of their interaction with Fcg receptors, the pathogenic potential of modified LDL-IC is unquestionable. Furthermore, clinical studies carried out in patients with type 1 diabetes have clearly demonstrated that high levels of modified LDL-IC predict cardiovascular events. Our observations have also additional clinical implications. On one hand, we have demonstrated that the assay of modified LDL and corresponding antibodies in serum is extremely inaccurate, due to the interference of IC. On the other hand, our studies suggest that measurements of modified LDL in IC, rather than serum, are required for the evaluation of the pathogenic potential of modified LDL. In conclusion, the autoimmune response triggered by spontaneously modified LDL seems to be a significant factor in the pathogenesis of atherosclerosis. 1 1 Department of Medicine, Division of Immunology and Rheumatology, Stanford University, Stanford, CA, USA.Anergy can be induced in CD4+ T cell clones by a strong TCR signal in the absence of costimulatory signals. CD28/B7 costimu-lation has been shown to play a critical role in preventing anergy induction. Two models have been proposed to explain these results: one predicts that CD28 costimulation has a direct inhibitory effort on factors involved in anergy induction. The other suggests that CD28 costimulation results in the production of growth factors, such as IL-2, which in turn prevents anergy.We have identified GRAIL (gene related to anergy in lymphocytes) using differential display. GRAIL contains a highly conserved zinc binding ring domain and exhibits E3 ligase activity in vitro. Our studies have shown that constitutive expression of GRAIL, but not the enzymatically inactive form, H2N2 GRAIL, is sufficient to induce anergy in naRve CD4+ T cells, suggesting that GRAIL is an anergy factor. Furthermore, we have shown that pharmacological inhibition of downstream targets of CD28 signaling such as PI3K and mTOR, as well as blocking IL-2 signaling using an antagonist antibody, all result in a significant increase in GRAIL expression, indicating that GRAIL is regulated by CD28. Our results suggest that GRAIL functions to maintain anergy in T cells through the ubiquitination pathway to degrade essential signaling molecules that are involved in T cell immunity. Our studies also shed light on the signal transduction pathways downstream of CD28/IL-2 that are involved in GRAIL expression, thereby elucidating the basis of costimulation blockade. A better mechanistic understanding of GRAIL and its regulation by costimulatory molecules could lead to novel treatments for patients suffering from autoimmune disease or from transplantation complications. Background: Type1 diabetes (TID) is an autoimmune disease caused by the pathogenic action of T lymphocytes which destroy insulin producing beta cells. An effective immunomodulatory anti-CD3 antibody, hOKT3g1(Ala-Ala), used to treat new onset TIDM had direct effects on pathogenic T cells and induced regulatory cells. Methods: In a Phase II trial sponsored by the Immune Tolerance Network, we studied the effects of the hOKT3g1(Ala-Ala) on immune responses in patients with new onset TID. Six patients were treated with hOKT3g1(Ala-Ala) I.V. Immunophenotyping; T cell proliferation by CFSE-based flow cytometry; and cytokine secretion were performed. Results: Transient depletion of T cells in patients followed by recovery of the cells after the 12 day antibody course was observed in all but one patient who demonstrated a prolonged reduction in the number of circulating CD4+ T cells. Nine months after therapy, the patientTs CD4 count was 102Â10 6 /mL and did not return to normal levels till 29 months post therapy (512  10 6 /ml). In contrast, the CD8+ T cell count recovered rapidly and reached 86% of baseline by 30 days. No obvious difference in either coating or modulation of CD3 on T cells was observed on the CD4+ T cells as compared to the other treated individuals. Trough levels of drug were higher but not exceptionally different than observed in the majority of treated individuals. However, there was selective recovery of CD45RO cells and CD4+CD62L+CD25hi Treg population increased during the treatment. In spite of the reduced CD4+ T cell count, the T cells functioned normally on a per cell basis as demonstrated by CFSE proliferation and IL-2 secretion. Clinically, the patient had an uneventful clinical course with only a transient viral syndrome shortly after treatment, with all cultures and serological studies negative. The patient was maintained on antimicrobial prophylaxis until her CD4+ count recovered. The patientTs c-peptide levels remain high at 2 years (77% of baseline) while maintaining a normal Hemoglobin A1c at 18 months comparable to that noted in other responders. Discussion: In the present clinical study of hOKT3g1 (Ala-Ala) one patient experienced a prolonged decrease in CD4+ T cells. The therapy did not lead to impaired CD4 function ex vivo, or to opportunistic infections. The unremarkable clinical course may relate to lack of depletion centrally, and/or failure to cause more generalized immunosuppression. Since the higher circulating levels did not result in a better clinical response, the data support dosing at the previous lower phase I/II levels helping define the window for Daniel H. Li, 1 Albert Chen, 1 James Tung, 2 Paulo Fontoura, 3 Larry Steinman, 3 Jane R. Parnes. 1 CD72, a type II transmembrane protein expressed predominantly on B cells, acts as a negative regulator of B cell activation through the B-cell receptor (BCR). However, whether CD72 plays a role in autoimmunity is unknown. We found that aged CD72deficient mice develop lupus-like autoimmune features such as anti-nuclear Ab, anti-DNA Ab, and glomerulonephritis. To study the mechanism of this spontaneous autoimmune disease, we employed a transgenic mouse model of autoimmunity to elucidate how CD72-deficient B cells break down tolerance in vitro. B cells from double-transgenic (dTg) mice expressing an anti-HEL (hen egg lysozyme)-specific BCR and soluble HEL in the circulation are anergic (unresponsive) to HEL antigen (Ag) stimulation. We found that B cells from CD72 HEL dTg mice are hyperproliferative in response to HEL Ag stimulation, as compared to the anergic response of B cells from wild-type (WT) HEL dTg mice. To further understand the signal transduction pathways employed by CD72, the downstream signaling events such as NF-kB, NF-AT, ERK, p38, and JNK pathways were examined. The results show that B cells from CD72 HEL dTg mice have (1) greater NF-kB p65 DNA binding activity, (2) greater NF-ATc1 activation, (3) greater ERK activity, (4) greater JNK activity, and (5) greater p38 activity, as compared to those of B cells from WT HEL dTg mice. We found that the calcium flux in response to HEL Ag stimulation is greatly enhanced in CD72 HEL dTg B cell when compared to the WT HEL dTg B cells. To identify the targets of CD72 signaling immediately following stimulation, we examined the phosphorylation status of many early signaling molecules such as Lyn, Syk, Ig-a/b, PLCg-2, Blnk, Vav, and Cbl etc. To determine whether these effects of CD72 on B cell tolerance have clinical relevance to the development of autoimmune disease, we induced EAE (experimental autoimmune encephalomyelitis) in both WT and CD72KO mice. We found that MOG (35-55) peptide induced an earlier onset and more severe EAE in CD72KO mice as compared to WT mice. Taken together, these findings indicate that CD72 plays an essential role in modulating B cell peripheral tolerance.Su1.72. Identification of the Target Self-Antigens in Ischemia/Reperfusion Injury. Ischemia/reperfusion injury (I/R), a potential life-threatening disorder, represents an acute inflammatory response following periods of ischemia resulting from myocardial infarction, stroke, surgery or trauma. The recent identification of a monoclonal natural IgM that initiates I/R led to the identification of conserved selfantigens as the target. Utilizing proteomics and phage-displaypeptide library, we have identified the antigenic epitope of I/R specific antigen and developed peptide inhibitors for injury in three different models (intestine, skeletal muscle, and heart). These results identify a novel pathway in which the innate response to a highly conserved self-antigen results in tissue destruction through a generalized autoimmune mechanism. Our previous studies revealed common HLA class II haplotypes that confer predisposition or protection to IDDM, MS, JCA, pemphigus vulgaris (PV) and SLE in the Bulgarian population. In order to evaluate further the role of HLA antigens in those autoimmune diseases we looked for common sequence motifs in the hipervariable regions of molecules encoded by protective and predisposing DRB1 and DQB1 alleles. We found that the b-chains coded by the common predisposing DRB1*0301, 0401, 0402, 0404 alleles have similarities in HVR III : a motif 57D58A in pocket 9 and an electric charge of pocket 4, generated by residues 70-74. Similar characteristics are observed for molecules coded by other predisposing DRB1 alleles in our population: 1401 and 1404 (PV); 1501 and 1001 (MS); 0701 (SLE). Part of this motif (13G, 26L) is shared by other predisposing for our population alleles: DQB1*0602 (MS), 0604 and 0303 (SLE). The b-chain, encoded by the common protective DQB1*0301 is characterized by different, hydrophobic amino acids-13A, 26Y, and has D at position 57 similarly to DQB1*0503-another protective for IDDM allele in Bul-garians. In Vivo Blockade of Human IL-2 Receptor (IL-2R) Induces Expansion of CD56 bright Regulatory NK Cells in Patients with Active Uveitis.Zhuqing Li, 1 Wee Kiak Lim, 1,2 Sankaranarayana P. Mahesh, 1 Robert B. Nussenblatt. 1 1 Laboratory of Immunology, National Eye Institute, NIH, Bethesda, MD, USA; 2 Singapore National Eye Center, Singapore, Singapore.In vivo blockade of the human IL-2 receptor (IL-2R) by antibody has been used for immunosuppression in transplantation, therapy for leukemia and autoimmune diseases. In this study, we report that administration of a humanized IL-2R blocking antibody induced a 4-to 20-fold expansion of CD56 bright regulatory NK cells in uveitis patients over time. The induced CD56 bright regulatory NK cells from the treated patients exhibited the same phenotype as those from normal donors except that they had a lower level expression of CD161. In addition, patients with active uveitis had a significantly lower level of CD56 bright regulatory NK cells in the periphery as compared to normal donors (P b 0.01). The induction of the CD56 bright regulatory NK subset occurred prior to the onset of a clinically therapeutic effect. Our observation may have implications for the potential role of CD56 bright regulatory NK cells in autoimmune diseases and to IL-2R blockade therapy. The decline in immunocompetence with age is accompanied by a steadily increasing incidence of autoimmune diseases. A reduced thymic output, which is an important aspect of immunosenescence, will induce compensatory auto-proliferation. This process can lead to a premature senescence of T cells and contribute to the immune abnormalities associated with autoimmunity and aging. In this study we tested whether the immune system of patients with rheumatoid arthritis (RA), multiple sclerosis (MS) and ankylosing spondylitis (AS) shows signs of an age-independent aging. Therefore, we measured two indicators of aging, the number of T cell receptor excision circles (TRECs) and the percentage of CD4+CD28 null T cells, in peripheral blood mononuclear cells (PBMC). In addition, characteristics of senescent CD4+CD28 null T cells were analyzed. PBMC were isolated from blood of 60 RA, 30 MS and 20 AS patients as well as 40 healthy controls (HC). The percentage of CD4+CD28 null T cells was measured by flow cytometry. The number of TRECs in 100 ng of gDNA was determined by real-time PCR analysis. Intracellular FACS analysis and CDR3 fragment length analysis were used to determine the cytokine profile and the clonal origin of the CD4+CD28 null T cells, respectively. In RA and MS patients, TREC numbers were significantly decreased compared to age-matched HC. TREC numbers were comparable between AS patients and HC. Furthermore, an increased percentage of CD4+CD28 null T cells was detected in 40% of RA patients, 32% of MS patients and 12% of AS patients versus 10% of HC. In HC and RA patients, the presence of the HLA-DR4 haplotype, a genetic risk factor for developing RA, was found to be linked with the percentage of CD4+CD28 null T cells. CD4+CD28 null T cells were also detected in the synovial tissue of RA patients. Analysis of the CD4+CD28 null T cells revealed clonal expansions and pro-inflammatory properties. This study provides indications for a premature senescence of the immune system in both RA and MS patients. Premature aging, associated thymic involution and compensatory auto-proliferation might play an important role in the pathogenesis of autoimmunity. CD4+CD28 null T cells have tissue damaging properties, are prematurely increased in both RA and MS patients and might therefore play an active role in the pathogenesis of these autoimmune diseases. Further study will be necessary to reveal the exact role of an aged immune system and more particular the role of CD4+CD28 null T cells in the pathogenesis of autoimmunity. It has been shown the presence of autoantibodies in patients with thyroid immunity disease and IgG, IgA and IgE antibodies in patients with Helicobacter pylori infection.Chronic bidiopathicQ urticaria and cough are present in almost 50% of patients with either diseases. In a subset of chronic b idiopathicQ urticaria patients, the disease is caused by the presence of IgG autoantibodies against the high affinity IgE receptor FcqRIa and anti-IgE autoantibodies. These functional autoantibodies stimulate normal activation of mast cells and basophils via FcqRIa causing whealing, angioedema and cough.The aim of the study is: to evaluate in patients with chronic idiopathic urticaria and/or cough: the presence of Helicobacter pylori antibodies and/or thyroid autoimmunity the histamine release and the prevalence of IgG autoantibodies against FceRIa and anti-IgE autoantibodies in patients positive for thyroid autoimmunity or Helicobacter pylori infection before and after levothyroxine sodium or HP eradication therapy Methods All patients had chronic urticaria and /or cough, for all of them the following investigations were performed: skin prick test for aeroallergens and food allergens, blood test for antithyroid antibodies (AAT, ATPO), FT3, FT4, TSH., Helicobacter pylori antibodies (IgG, IgA), total IgE, autologous serum skin test (ASPT) in patients with positive anthithyroid and HP antibodies, histamine-release activity, serum IgG anti-Fca RI e and anti-IgE in all patientsT sera Results Our preliminary data show: 100% of patients were with normal thyrotropin levels 100% of patients with thyroid autoimmunity had cough and 33% of them also had urticaria 100% of patients with HP positive had only urticaria and 41% of them also had cough 85% of the patients had ASPT and antibodies positive, 80% had ASPT and histamine release positive 70 to 100 % remission rate (either partial or complete) of urticaria and cough in patients treated with levothyroxine.All patients treated with HP eradication therapy had negative breath test after treatment; 80% of them had remission of urticaria. 85% of the patients had ASPT and antibodies positive, 80% had ASPT and histamine release positive Conclusions These results suggest that histamine releasing autoantidodies are important in the pathogenesis of chronic urticaria and cough by stimulating or facilitating degranulation of basophils and coutaneous mast cells through cross-linking cell surface IgE receptors. Receptors for the constant region of IgG (FcgRs) link the humoral and cellular arms of the immune system. Their dysregulation has been implicated in the development of autoimmune conditions particularly systemic lupus erythematosus (SLE). A polymorphism at position 232 in the transmembrane domain of the inhibitory receptor, FcgRIIb, results in an isoleucine to threonine substitution is prevalent in SLE patients. Using the FcgRIIbnegative monocytic cell line, U937, we assessed the consequences of expression of either wild type (wt) or mutant (232T) FcgRIIb on activatory Fcg receptor function. Expression of wt but not 232T prevents lysosomal trafficking of immune complexes and superoxide generation, inhibits phagocytosis of opsonised pneumococci and reduces pro-inflammatory cytokine release. The 232T receptor is unable to partition into lipid rafts, excluding it from the proinflammatory signalling complex. Primary macrophages from 232T homozygotes show similar defects in FcgR control, correctable by lentiviral introduction of wt FcgRIIb. Thus, this receptor polymorphism radically alters the cellular response to opsonised particles and may predispose to the development of SLE. This mutation is the first described which could contribute to disease by excluding a surface receptor from lipid rafts, potentially representing a novel mechanism underlying human genetic disease. THE AIM OF INVESTIGATION The aim of the investigation was the study and compare of in vitro synthesis and serum concentrations of gIFN, IL18 and TGFb 1 in patients with AITG. THE MATERIALS AND METHODS 25 patients with AITD were studied. The control group consisted of 12 healthy volunteers with similar to the investigated patients in sex and age. The serum concentrations of gIFN, IL18 and TGFb 1 and MNC supernatant levels (24-48 h culture) of cytokines were determined using ELISA method. RESULTS An increase in the serum concentrations of both cytokines (gIFN, IL18) and growth factor TGFb 1 were noted in the studied group when compared to the control group. The increase in serum concentrations of gIFN, IL18 accompanied by their increased synthesis by peripheral MNC more than 2.5-3.2 times in comparison to control data. The average serum concentration of TGFb 1 in AITD was 274 pg/ml and in control group-47.4 pg/ml (P = 0,02). The serum concentrations of gIFN and IL18 were from 3.8 to 4.5 times higher than in control group.CONCLUSION The increase of the serum concentrations of gIFN, IL18 and TGFb 1 and increased synthesis by peripheral MNC of gIFN and IL18 suggest that Th1 cytokines as well as growth factor TGFb 1 plays an important role in AITD. Autoimmunity: Common Origin for Diverse Diseases. 1,2 1 Rheumatology, Clinica Universitaria Bolivariana, Medellin, Colombia; 2 Cellular Biology and Immunogenetics Unit, Corporacion para Investigaciones Biologicas, Medellin, Colombia.Objective: The current study investigated the presence of autoimmune diseases (AID) among first degree relatives (FDR) of primary SjfgrenTs syndrome (pSS) and type 1 diabetes mellitus (T1DM) patients. Materials and Methods: To this purpose, a case-control study was undertaken in which individuals were interviewed personally using a questionnaire that sought information about demographic and medical characteristics including AID family history. Results: In pSS, there were 101 women defined according to the Revised European Criteria and 124 matched controls without AID. We found that 38 (38%) families had at least one FDR having an AID, while in controls there were 27 (22%) (OR: 2.2, 95%CI = 1.2-3.9, P = 0.01). An AID was registered in 56 (6%) of 980 FDR of patients as compared with 33 (2%) of 1433 FDR in controls (OR: 2.6, 95%CI = 1.66-3.98, P b 0.0001). Sixtyfour AID were observed in the 56 FDR of patients, of whom five had more than one AID. In patients with T1DM, defined according to the Expert Committee Criteria, there were 107 patients and 113 matched controls without AID. In patients, there were 25 (26%) FDR with at least one AID while in controls there were 9 (8%) with the same characteristics (OR: 3.96, 95%CI = 1.74-9, P = 0.0006). Among the T1DM patients, an AID was registered for 26 (8.3%) of the 312 FDR as compared with 9 (2.4%) of the 362 FDR among controls (OR: 3.56, 95%CI = 1.64-7.32, P = 0.0008). The most frequents AID registered in FDR of pSS patients were autoimmune hypothyroidism and rheumatoid arthritis, and in FDR of T1DM patients were autoimmune hypothyroidism and T1DM. Conclusion: These results indicate that familial autoimmunity is a major trait in AID, thus sustaining the common variants/multiple disease hypothesis which emphasizes that similar immunogenetic mechanisms underlie AID. In experimental autoimmune uveoretinitis (EAU), an animal model of autoimmune inflammatory eye disease, activated macrophages play a critical role in tissue destruction in the retina, since depletion of macrophages reduces retinal damage without concomitant reduction in numbers of other infiltrating cells. Macrophage activation is modulated by signals received in the local micro-environment from soluble cytokines such as IFNg and TNF-a, as well as signals received from cell surface receptor interactions, for example of CD200 with CD200R. Using NOS2 mediated production of nitrite (NO) as a measure of activation in vitro and EAU to study the course of disease in vivo, we have analysed the activation of wild type or TNFR1 -/macrophages.As in the model of multiple sclerosis, experimental autoimmune encephalomyelitis, TNFR1 -/mice are resistant to the induction of EAU. They have a lower incidence of disease and lower disease scores. Priming of T cells is only modestly impaired, but by the time the animals reach peak disease (day18-21), antigen specific proliferation in the spleen is significantly reduced in the TNFR1 -/mice. To investigate this further macrophage function was assessed in vitro by activation with various stimulators. In response to stimulation with IFN-g, wild type mice produce substantial amounts of NO. On the other hand TNFR1 -/mice cannot be stimulated to produce NO by IFN-g activation. To test whether this was due to autocrine stimulation by low levels of TNF-a, we stimulated wild type macrophages with IFN-g in the presence of sTNFR-Ig fusion protein, which binds free TNF-a, and showed that this also blocked NO production. In contrast with this impaired activation of NO synthesis, MHC class II upregulation was indistinguishable in wild type and TNFR1 -/mice stimulated with IFN-g. We then addressed whether TNFR1 signalling was essential for NO production, by stimulating macrophages with LPS. Under these conditions both wild type and TNFR1 -/mice produced equivalent amounts of NO.Together these results show that selected aspects of IFN-g mediated activation of macrophages are controlled by autocrine secretion of TNF-a, but that this control is bypassed in the presence of signals generated by pathogen-associated molecular pattern recognising receptors. Macrophage activation in a proinflammatory milieu may therefore be modified by the presence of pathogen derived signals, and may be different in autoimmune disease where inflammation occurs in the absence of foreign immune system activators. This implies that in this model, activation sufficient for priming occurs in the presence of mycobacterial derived products in draining lymph nodes, but not when macrophages are exposed to IFN-g alone in the retina. CD4CD25+ regulatory T cells (Tregs) , are fundamental to the maintenance of peripheral tolerance and have great therapeutic potential. However, efforts have been hampered by limiting cell numbers in vivo, an anergic phenotype in vitro, and only a rudimentary understanding of the molecular basis for the functional state of CD4CD25+ Tregs. We have previously shown that T cell activation through the TCR results in the activation of CD44 to bind its ligand hyaluronan (HA), and that the CD44/HA interaction is used for T cell extravasation at sites of chronic inflammation. Here we show that CD4CD25+ cells more rapidly and extensively activate the functionally active, hyaluronan-binding form of CD44 (CD44 act ) after TCR triggering, with~85% of cells expressing this marker compared to~25% of CD4+CD25-cells after 40 hrs of activation. Moreover, CD44 act expression is strikingly correlated with suppressor activity in CD4CD25+ T cells, in contrast to other surface markers or Foxp3 expression. Within 16 hr after in vitro activation, CD44 act can discriminate enhanced suppressive function in in vitro proliferation assays and in vivo models. After separation of CD4CD25+ Treg into HA-binding and non-HAbinding fractions, multiple cytokines, including IL-4, IL-10, and TGF are preferentially expressed by the HA-binding cells. These cells also show the preponderance of suppressive function in vitro and in two in vivo models of allogeneic GVHD. CD44 act is induced on resting CD4CD25+ cells in vivo with antigenic stimulation, with similar functional consequences. These results reveal a cell surface marker that delineates functional activity among CD4CD25+ regulatory T cells, thereby providing a novel tool to aid in identifying regulatory activity and enriching for maximal suppressor potency. The main feature of autoimmune disease such as autoimmune inner ear disease including MeniereTs disease is the development and persistence of inflammatory processes in the apparent absence of pathogens, leading to destruction of the target tissues. Western blot analysis has shown that 59% of MeniereTs disease patients produce antibodies to a 55 kD inner ear membranous and neural protein identified to be h-tubulin. Hearing loss in mice can be induced by immunization with h-tubulin using ABR and DPOAE recorder and spiral ganglion as well as hair cells damages can be observed by morphological study of temporal bones. But the precise immunological mechanism of inner ear disease remains obscure.In the current study, balb/C mice were subcutaneously injected with h-tubulin in dosage of 100, 200 and 300 Ag with CFA per mouse, immunizations were boosted in IFA with varying doses of tubulin twice at one-week intervals. Control mice underwent subcutaneous injection of PBS and CFA/IFA as well. After 2 weeks of last boosting, we have found that the antibodies activity to htubulin increased in dose dependent compared with controls, all control subjects were relatively unresponsive. Moreover, IFNgamma level was markedly increased in both serum and supernatant of lymphocytes, protein levels of TGF-h, IL-4, IL-5 IL-10 and IL-13 were significantly reduced following h-tubulin immunization. Interestingly, flow cytometric analysis of spleen cells from h-tubulin induced mice and control mice have been showed that 2.72% of total splenocytes in control mice were CD25 + CD4 + regulatory T cells (Treg cells), the population of Treg cells was reduced in h-tubulin induced mice and followed dose dependent (such as 1.68% in 300Ag, 2.16% in 200 Ag and 2.19% in 100 Ag), the Treg cells in naRve mice is 2.6%. Moreover, IFN-gamma and IL-2 level was markedly increased in supernatant of Treg cells culture, protein levels of TGF-h, IL-4, IL-5, IL-10, IL-12p40 and IL-13 were significantly reduced following h-tubulin immunization.Thus, these data indicate an immune reactivity against htubulin, which might be responsible for the autoimmune inner ear hearing loss. The further study is required to elucidate the role of CD25 + CD4 + T cells in the pathogenesis of this disease, which would eventually result in better therapy. bNaturalQ CD4+CD25+ regulatory T cells (T-reg) develop in the thymus, apparently as a result of interaction with their cognate antigen. We previously showed that susceptibility to experimental autoimmune uveitis (EAU), that is elicited in mice with the retinal autoantigen IRBP and serves as a model for human uveitis, is controlled by thymus-derived T-reg. In this study we examine whether endogenous expression of IRBP is necessary to generate EAU-relevant T-reg, by comparing IRBP knockout (KO) and wild type (WT) mice. We hypothesized that, if endogenous IRBP is needed to generate EAU-relevant T-regs, depletion of CD25+ cells from IRBP KO mice will not alter their responses to IRBP. Mice were depleted of CD25+ cells with the monoclonal antibody PC61. To induce EAU, mice were immunized with IRBP in complete FreundTs adjuvant (CFA), or were infused with T cells from IRBPimmunized donors. Some mice were immunized with IRBP in incomplete FreundTs adjuvant (IFA), a non-uveitogenic regimen. EAU scores and associated immunological responses (delayed hypersensitivity and proliferation to IRBP) were examined. When immunized with IRBP/CFA, CD25 depleted WT and KO both had enhanced immune responses to IRBP. Furthermore, IRBP primed T cells of CD25 depleted KO donors elicited more severe disease in WT recipients than did cells from non-depleted KO donors, suggesting that an EAU-relevant T-reg had been removed. However, when immunized with IRBP/IFA, only the depleted WT, but not the depleted KO mice, had enhanced IRBP-specific immune responses, suggesting that IRBP specific T-regs were present only in WT. We conclude that, since endogenous expression of IRBP appears to be needed to generate IRBPspecific T-regs, the EAU-relevant T-regs in IRBP KO are not IRBP-specific. We propose that these are T-regs activated by microbial components present in CFA, that inhibit development of IRBP-specific effector T cells in a bystander fashion. Thus, the innate immune stimulation that is needed to elicit EAU also activates regulatory T cells that help control the disease. Background: Current treatment options for autoimmune conditions are unable to specifically prevent or stop the autoimmune process. Therefore, there is a need for new antigen specific therapies for autoimmune diseases. We evaluated the efficiency of a new APL technology referred to as Ligand Epitope Antigen Presentation System (L.E.A.P.S.k) to prevent or treat experimental autoimmune myocarditis (EAM). J-My-1 is a conjugate of My-1 peptide from the a chain of murine cardiac myosin, which is used to induce EAM, and J peptide that is a part of the human h-2 microglobulin molecule.Methods: To evaluate prophylactic or therapeutic vaccination, 6-8 weeks old female A/J mice were injected with J-My-1 emulsified in ISA-51 adjuvant on days-14 and-7 prior to EAM induction and sacrificed on day 28 (prophylactic), or injected on days 0, 7, 14, 21 following EAM induction and sacrificed on day 28 (therapeutic). EAM was induced by immunization with My-1, emulsified in CFA on days 0 and 7 and injection of pertussis toxin on day 0. Control groups were vaccinated with PBS emulsified in ISA-51, using the same schedule. Experiments were repeated at least twice with 8-10 mice per group. Myocarditis severity was evaluated by histology and by the heart / body weight ratio. My-1 specific antibodies (total, IgG1 and IgG2a) were analyzed by ELISA. Th1 and Th2 cytokines were evaluated by Linco-Multiplex (for 22 cytokines) from sera as well as from heart and spleen homogenates by ELISA. To evaluate whether the mechanism of J-My-1 action was due to general anergy or specific response towards My-1 peptide, we examined serum antibodies against mycobacterial antigens (PPD) in the CFA by ELISA and antigen (My-1) specific lympholiferation in vitro.Results: J-My-1 administration reduced the incidence and severity of EAM when given before or after the EAM induction. In both cases, the J-My-1 treatment significantly lowered the heart/ body weight ratio (prophylactic, P = 0.047; therapeutic, P = 0.006), severity of myocarditis assessed by histology (prophylactic, P = 0.0187; therapeutic, P = 0.002) and lower levels of anti-My-1 antibodies. The decreased immune response was antigen specific, as J-My-1 had no effect on antibody responses to mycobacterial antigens. Spleen lymphocytes from J-My-1 treated mice had reduced proliferative responses to My-1. The severity of EAM was not reduced with My-1 and ISA51 in contrast to J-My-1. altered peptide ligand as a preventive vaccine as well as a treatment, we were able to significantly reduce incidence and severity of experimental autoimmune myocarditis. As a result of such treatment, we observed increased levels of IL-13 (which we found to be protective) and levels of proinflammatory IL-1h in sera and IFNg in heart decreased. Activation of self-reactive T cells in healthy adults is prevented by the presence of autoantigen-specific CD4 + CD25 + regulatory T cells (CD25 + Treg). To better understand where human autoantigen-reactive CD25 + Treg are selected and expanded we investigated if thymic CD25 + Treg from children and CD25 + Treg from cord blood are able to suppress proliferation and cytokine production induced by specific antigens. CD4 + CD25-and CD4 + CD25 + cell fractions where purified by magnetic cell separation and proliferation and cytokine production where analysed after stimulation with the neural antigen myelin oligodendrocyte glycoprotein (MOG) and the polyconal stimuli staphylococcal enterotoxin B (SEB). CD4 + CD25-thymocytes proliferated in response to MOG but the addition of an equal number of thymic CD25 + T cells did not significantly suppress this proliferation. However thymic CD25 + T cells inhibited IFN-g production induced by MOG, which indicates the presence of MOG-responsive CD25 + Treg in the thymus. In contrast, cord blood CD25 + Treg suppressed both proliferation and IFN-g production induced by MOG. Interestingly, thymic but not cord blood CD25 + T cells suppressed proliferation and cytokine production induced by SEB. Both cord blood and thymic CD25 + Treg expressed FOXP3 mRNA, but FOXP3 expression was lower in cord blood than in thymic CD25 + T cells. This is probably due to the presence of CD45RA-FOXP3 low cells in addition to CD45RA + FOXP3 high cells in cord blood. In summary, suppression of immune responses to MOG where more easily detected in cord blood than in thymus which, suggests that MOG-reactive CD25 + Treg are further expanded in the periphery. However, cord blood derived CD25 + T cells did not suppress SEB induced responses and expressed lower levels of FOXP3, which indicates that all CD25 + T cells in cord blood are not regulatory. Antigen processing in the MHC II compartment of human dendritic cells (DC) is poorly understood, however, strategies that might interfere with the processing of e.g. In particular, information about the nature and the sequence of proteases, especially cathepsins (cat), attacking a given exogenous antigen after uptake by live human DC is lacking. Using a similar approach, we have here followed the uptake and cathepsin-binding of such affinity probes by live human DC generated from monocytes in vitro. We found that, in contrast to murine DC, human DC only very poorly internalize exogenous material by phagozytosis, but are much more efficient in fluid phase-endocytosis-mediated uptake of the probe, compared to murine cells. While in cell lysates active catS, catB, catH, catZ and catL were decorated by the probe, internal-ization of the probe by live human DC revealed only a selective labelling of catS and catB, but no decoration of catH and catZ by endocytosis. In a pulse chase analysis, the signal for active catS selectively increased over a chase period of 60 min, indicating that exogenously added material was selectively routed to catScontaing compartments after fluid phase endocytosis in human DC. Treatment of DC with chloroquine reduced cellular uptake and cathepsin labelling, as expected, while the H+-ATPaseinhibitor Concanamycin B selectively abrogated labelling of CatB, most likely due to the pH-dependent activity of the enzyme. Treatment of DC with LPS not only reduced the total amount of catS reached by the probe due to decreased internalization, but also abrogated the increase in catS labelling that was seen in untreated DC without a maturation stimulus, while the total amount of active catS remained unchanged. This suggested that not only internalization, but also the intracellular transport to catScontaining compartments is affected by DC maturation. In summary, catS is liekly to present the functionally dominant cysteine protease for antigen processing in the MHC class II compartment of live human DC, because exogenous pan-cathepinreactive probes selectively bind to catS after internalization by fluid-phase endocytosis. T Cell Responses Induced by Myelin Su1.87. Objective of the study: CD4 + CD25 + regulatory T cells (Treg) suppress T cell activation in vitro and regulate multiple immune reactions in vivo. Here, we show that IL-2 is in addition an important activator of Treg suppressive activity. Moreover, since Treg do not produce IL-2 by themselves but depend on IL-2 produced by activated T cells we also provide a new concept for the regulation of Treg activity by there own target cells via IL-2.Materials and methods: We used in vitro cultures systems of purified T cell populations to analyse the specific effect of IL-2 on Treg. In addition we studied the role of IL-2 for Treg activity in an in vivo T cell transfer system.Results: We and others have previously shown, that in vitro blockade of the IL-2 receptor on Tregs during co-culture with responder T cells completely blocks their suppressive activity. Here we demonstrate, that the uptake of IL-2 during priming of Treg is required to induce the capacity to produce IL-10 upon restimulation. IL-4 which induces significant production of IL-10 in naRve T cells is not sufficient to induce IL-10 production from Treg but acts synergistically with IL-2. Furthermore, in vivo application of IL-2 by gene-gun immunization in normal mice selectively activates Treg and induces the complete suppression of proliferation of a transferred population of Ova-specific T cells in response to vaccination with ovalbumin.Conclusion: Our results show that IL-2 is a potent inducer of Treg suppressive activity including the production of IL-10. The regulation of Treg activity by IL-2 produced from their own target cells allows to adapt Treg effector function to the strength of an immune response. Furthermore, the specific activation of Treg suppressive activity in vivo may represent a new strategy for the therapeutic intervention in autoimmunity and chronic inflammation. Natural antibodies as well as antibodies from an autoimmune strain of mice (B6.MRL/lpr) have been shown to mediate mesenteric ischemia/reperfusion (I/R)-induced tissue injury. Intestinal injury is initiated when neoantigens reveled on ischemic or apoptotic cells are bound by antibody and complement is activated. In addition to the local (intestinal damage), remote tissue damage in the lung occurs. In the autoimmune mice it has been reported that injury in older (five month) animals is accelerated with increased severity. Passive antibody transfer, of either purified natural antibody or IgG serum from older B6.MRL/lpr mice, into Rag1 deficient mice restores I/R-induced tissue damage. These previous studies indicate that auto-antibodies contribute to tissue damage through complement activation after a tissue or organ suffers from another damaging event such as ischemia. However it is not clear if a particular neoantigen(s) is dominant in initiating the I/R-induced tissue damage. Nor is the relationship between local (intestinal) and remote (lung) tissue damage clear. Subsequently, we have expanded upon the autoimmune studies to examine if autoantibodies with specificities for either dsDNA or ssDNA at varying concentrations would cause equivalent local and remote I/Rinduced tissue damage. As expected, at high concentrations, the passive transfer of all anti-DNA antibodies into Rag1 -/-mice resulted in local intestinal and remote damage. However at lower concentration the anti-dsDNA antibody was more efficient in facilitating mesenteric I/R-induced tissue damage. When remote lung damage was evaluated the transfer of the anti-dsDNA antibody resulted in increase septal thicken and cellular infiltrate in the lung compared to the transfer of anti-ssDNA antibodies or from wild type control mice. Further studies addressing the recruitment of cellular infiltrate, levels of complement deposition, and the signals controlling polymorphonuclear neutrophil infiltration into intestinal or lung tissue are ongoing. These studies may help clarify the role of circulating auto antibodies in organ damage in patients and mice with systemic lupus erythematosis. CD48 is a member of the CD2 superfamily of costimulatory molecules and has an important role in regulating the immune response. CD48 is broadly expressed on B cells, T cells, dendritic cells, macrophages and NK cells, and may indicate that this molecule participates in immune regulation via various cell types. The importance of the interaction between CD48 and its receptors, CD2 and CD244 (2B4), in autoimmunity has not been clearly addressed. Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by production of autoantibodies, in particular anti-nuclear antibodies, B cell hyperactivity and activation of self-reactive T cells. Genetic studies indicated that three regions on different mouse chromosomes (SLE 1,2 and 3) are linked with susceptibility to the SLE at different severities. A subregion of the SLE 1 locus on mouse chromosome 1 (SLE1b) includes members of the CD2 superfamily The present study investigated the role of CD48 in tolerance and autoimmunity by studying the spontaneous autoimmune disease that occurs in aging CD48-/-mice. In CD48-/-mice, anti dsDNA IgG antibodies levels were higher than IgM but levels of both were at least 2-fold higher in 6-month old compared to 3-month old mice. Phenotypic analysis of T cells in 6-month old mice showed that levels of activation markers including CD62L, but also CD69 and CD25 were increased at least 2-fold on CD4 and CD8 T cells of CD48-/-mice compared to their WT counterparts. By T cell cloning, we have demonstrated the presence of anti-nucleosomal T cell responses in CD48-/-mice. The breakdown in tolerance is not a result of a defect in activationinduced cell death therefore further studies are required in order to delineate the mechanism underlining the regulatory effect of the CD48 pathway. Overall, the present study presents evidence suggesting that CD48 plays a key role in the control of autoimmunity. A small population of CD4 + T cells present in both humans and mice has the ability to regulate immune responses. These cells, known as regulatory T cells (T R ) are characterized by the expression of CD25 and FoxP3. Originally, CD4+CD25+ T R were thought to be derived from the thymus. Recent data suggests that adaptive CD4+CD25+ T R can be generated in the periphery. In a previous report, we have shown that activation of human CD4+CD25-results in expression of FoxP3 and CD25, as well as, acquisition of a cell contact dependent, cytokine independent regulatory activity by a portion of the original CD4+CD25-T cells. Recently, we have focused on determining the factors involved in generating this type of T R and their lifecycle. Results from our lab show that the addition of IL-2 to generation cultures results in an increased number of resulting regulatory T cells, whereas, blockade of the cytokines TGF-h and IL-10 by the addition of blocking antibodies to the generation culture has no effect on the number or function of the resulting T R . This demonstrates that the generation of these T R is independent of TGF-h or IL-10 and is enhanced in the presence of IL-2. Furthermore, results show that the CD4+CD25+FoxP3+ T R resulting from the generation cultures will retain their suppressive ability upon restimulation and when reactivated in the presence of high dose IL-2 can be expanded 50fold. Together these results suggest that generated T R may be a good candidate for immunotherapy due to their ability to retain suppressive activity and their capacity to be expanded. siRNA Targeted Reduction of IL-23 Production by Human Dendritic Cells Increases IL-10 Production and Decreases Antigen Presentation Capacity. A. Vaknin-Dembinsky, H. L. Weiner. 1 Neurology, Center for Neurological Diseases, Boston, MA, USA.Background: IL-23 is a heterodimeric cytokine that comprises a p19 subunit that associates with the IL-12/23p40 subunit. Like IL-12, IL-23 is expressed predominantly by activated dendritic cells (DCs) and phagocytic cells and both cytokines induce IFNsecretion by T cells. Unlike IL-12, IL-23 also functions by stimulating secretion of the pro-inflammatory cytokine IL-17 from T cells. A central role for IL-23 in autoimmune inflammation is supported by the observation that induction of experimental autoimmune encephalitis (EAE) occurred in mice lacking IL-12, but not in mice with targeted disruption of IL-23 or both IL-12 and IL-23. Thus IL-23 expression in dendritic cells (DC) may play an important role in the pathogenesis of human autoimmune diseases. One of the ways to explore the role of IL-23 in DC function is by down regulating DC gene expression.The aim of this study was to investigate the role of IL-23 in DC function and to evaluate the use of IL-23 specific antisense oligonucleotides (AS-oliges) and small interfering RNAs (siRNA) as a tool to silence IL-23 expression.Methods: We transfected human monocyte derived-DCs (mo-DCs) with specific AS-oligos to IL-23p19, IL-12 /23p40 and IL-12p35 and small interfering RNAs (siRNA) to IL-23p19. We compared the surface markers by FACS analysis and the APC function of mo-DCs that secrete lower amount of IL-23 and /or IL-12 to mo-DCS transfected with control AS-oligoes and control siRNA.Results: We found that mo-DCS are efficiently transfected with AS-oligos and siRNA (90-60%, respectively). Transfection of DCs with the AS-oligos and siRNA specific for the IL-23 p19 and IL-12 p35 genes resulted in potent suppression of gene expression and blockade of bioactive IL-23 and /or IL-12 p70 production without affecting unrelated genes or cellular viability. Inhibition of IL-23 and IL-12 was associated with increased IL-10 production and decreased TNF alpha production. Furthermore, transfected mo-DCs were poor allostimulators in a mixed lymphocyte reaction.Conclusions: We demonstrate that AS-oligos and siRNA can be used for immune modulation by targeting human dendritic cell (DC) gene expression. Targeted reduction in the expression of either IL-23 and /or IL-12 resulted in DCs that are less potent APCs and that secrete more IL-10. Knocking down the expression of IL-23 by AS-oligos or siRNA may be used as an avenue to develop therapy that target DCs in chronic inflammatory autoimmune diseases. Background: In a previous study, we found that the severe form of EAM in A/J mice exhibits a Th2-like phenotype. Blocking IL-4 with anti-IL-4 monoclonal antibodies (mAb) reduced the severity of EAM in A/J mice, showing that IL-4 plays a pathogenic role in EAM. We therefore speculated that another important Th2 cytokine; IL-13; could have an additive effect to IL-4. Methods: 6-8 weeks old IL-13 knockout mice (KO); IL-4 KO; IL-4/IL-13 double knockout (DKO) on BALB/c background and BALB/c wild type (WT) were immunized with myosin BALB/c peptide emulsified in CFA on days 0 and 7, injected with pertussis toxin on day 0. Myocarditis severity was evaluated by histology and by the heart/body weight ratio (HW/BW). Myosin peptide specific antibodies were analyzed by ELISA. Th1 and Th2 cytokines were evaluated from heart and spleen homogenates by ELISA. Results: Absence of IL-13 in IL-13 KO mice as well as in IL-4/ IL-13 DKO resulted in a significant increase in myocarditis incidence and severity assessed by histology and the HW/BW ratio. IL-13 KO mice had a higher histology score (P = 0.0000001) as well as HW/BW ratio (P = 0.00008) compared to BALB/c WT. IL-13/IL-4 DKO mice had still greater EAM severity than BALB/c WT mice; however, their phenotype was intermediate between IL-13 KO and BALB/c WT. It was challenging to show clearly that IL-4 is a pathogenic factor in BALB/c EAM as we observed in A/J EAM. BALB/c are moderate responders to the EAM induction; therefore we could not show a statistically significant lowering of myocarditis severity by histology in absence of IL-4 on BALB/c background. However, the HW/BW ratio was significantly lower in IL-4 KO mice than in BALB/c WT (P = 0.025), suggesting that IL-4 promotes disease in BALB/c mice. Next to increased EAM severity, IL-13 KO mice showed significantly higher levels of anti-myosin peptide IgG, IgG1, IgG2a and IgG2b compared to BALB/c WT. Discussion and Conclusions: By inducing EAM in IL-13 KO mice, we were able to significantly increase the incidence and severity of myocarditis, showing a protective effect of IL-13 in EAM in BALB/c mice. The protective effect of IL-13 was also observed in IL-4/IL-13 DKO mice, in which myocarditis severity was intermediate between IL-13 KO and BALB/c WT mice. This intermediate phenotype, together with the reduced disease in IL-4 KO and BALB/c with IL-4 blocked by anti IL-4 mAb, suggests a pathogenic role of IL-4 in EAM in BALB/c mice. The protective effect of the IL-13 is associated with increases of two key regulatory cytokines (IL-1 h and IFN-g) in a heart homogenate from IL-13 KO mice. The two main Th2 cytokines, IL-4 and IL-13, thus have opposite effects on EAM, IL-13 being a major protective factor and IL-4 being a pathogenic factor. Costimulation via the CD28/B7 pathway is required for activation of naive T cells; however, the precise role of this costimulatory pathway in memory T cell function has not been elucidated. In this study, we directly tested the role of the CD28/B7 costimulation pathway on CD4 memory function, by assessing the ability of CTLA4-Ig to block memory CD4 T cell function and expansion in vitro and in vivo. We used antigen specific CD4 T cells derived from transgenic mice expressing a transgene encoded T cell receptor specific for Influenza Hemagglutinin (HA) peptide (110-119) and I-E d and generated HA-specific memory cells by in vitro priming with HA and antigen presenting cells followed by adoptive transfer of HA-activated cells into syngeneic RAG2 -/hosts. HA-specific memory CD4 T cells were harvested from these adoptive hosts 8-10 weeks post transfer. In vitro recall of HAspecific memory CD4 T cells with HA in the presence of CTLA4-Ig led to a fifty percent reduction in the number of IL-2 producing cells, while IFN-g production was comparable between CTLA4-Ig and isotype control (IgG2a) treated cells. To test the effect of CTLA4-Ig on antigen stimulated HA-specific CD4 memory T cells in vivo, we transferred CFSE labeled HA-specific memory CD4 T cells into secondary BALB/c hosts and treated the recipient mice with CTLA4-Ig or IgG2a as an isotype control, then boosted these mice with HA peptide or PBS. We observed a reduced expansion of HA-specific CD4 memory T cells in HA-boosted recipient mice treated with CTLA4-Ig compared to IgG2a treated mice. The total antigen-specific memory CD4 T cell recovery of HA-boosted mice that received CTLA4-Ig versus control IgG2a was 20% of control in the spleen and 44% of control in the lymph nodes. These results indicate that the CD28/B7 pathway plays a key role in IL-2 production and in vivo expansion of re-stimulated memory CD4 T cells. These findings suggest a key role for CD28/B7 in promoting optimal memory T cell responses. Introduction: Complement C5a is a candidate target molecule for the treatment of inflammatory diseases such as respiratory distress syndrome and sepsis.The participation of C5a and C5aR in the development of autoimmune disease has not been investigated. The phagocytic responses by the two activating Fc receptors, FcgRI and FcgRIII, have been directly implicated in the pathogenesis of antibody-dependent autoimmune diseases, such as autoimmune vitiligo, immune thrombocytopenic purpura and autoimmune hemolytic anemia. In contrast, however, C5aR has not been thought to play a significant role in these diseases. Using an experimental model of autoimmune hemolytic anemia (AIHA) in C5aR-deficient mice, we show here that C5aR, although a nonphagocytic receptor, promotes cellular immune destruction in antibody-mediated autoimmune disease through a mechanism of bidirectional interaction between C5a and activating FcgR. Methods: Lethal AIHA was induced by a single intraperitoneal (IP) injection of aMRBC autoantibodies 34-3C. Kupffer cells were isolated by percoll gradient of the liver homogenate followed from control and anemic mice by sorting from the interphase by Mac-1coated magnetic beads. Surface protein was analysed by FACS and mRNA expression by Taqman RT-PCR. The liver supernatant was assayed for C5a-dependent chemotactic activity. Upon administration of anti-erythrocyte antibodies, C5aR deficiency resulted in impaired FcgR-mediated in vivo-erythrophagocytosis. The upregulation of the activating FcgR on Kupffer cells induced by the early C5a produced is also absent, in contrast to that observed in wild-type mice. Surprisingly, in mice deficient for FcgRI and FcgRIII, anti-erythrocyte antibody induced C5a production is abolished identifying activating FcgR as dominant mediators of autoantibody-induced release of C5a. Thus our data are consistent with a pathogenic mechanism of antibody-dependent hemolytic anemia in which IgG-opsonized erythrocytes trigger FcgR-mediated C5a release that in turn activates a positive cellular feedback signal uopn engagement of C5aR, which results in increased FcgRI and FcgRIII expression required for effective erythrophagocytosis. This shows that developement of a full blown antibody-dependent autoimmune disease requires co-operation of C5a and the activating FcgR in the same activation pathway and suggest therapeutic benifits of C5a-C5aR blockade in AIHA and other disease closely related to type II autoimmune injury.Work supported by the Deutsche Forschungsgemeinschaft (Ge 892/8-1).Su1.95. Regulatory T Cells: Differential Requirement for Innate and Adaptive Immune Cells Inhibition In Vitro.S. 1 1 Lymphocyte Physiology Unit, Instituto Gulbenkian de Ciencia, Oeiras, Portugal.CD4 + CD25 + regulatory T cells (T R ) have been implicated in the control of autoimmune diseases, allergy and inflammation. Development of in vitro model systems has facilitated the analysis of the functional properties of these cells. In this regard, inhibition of conventional T cell proliferation by T R is currently the most used assay. However, T R suppressor mechanism in these in vitro assays remains unknown and more importantly, appears to differ from the in vivo scenario. Whereas in the latter case, inhibitory cytokines, such as TGF-beta and IL-10, have been shown to play a role, this is not the case in vitro. In addition, while competition for IL-2 has been proposed to be the main suppressor mechanism in vitro, the presence of saturating amount of IL-2 in culture media was shown not to affect T R ability to suppress IL-2 production by the target cells. As similar results were obtained when testing Foxp3-negative suppressor cells, we conclude that IL-2 consumption by CD25-expressing T cells participates in a non-specific manner to the overall suppression of proliferation observed in these cultures. We next describe a suppression assay that does not rely on IL-2 consumption, as it monitors the inhibitory function of T R on components of the innate immune system. We show that Foxp3 + T R specifically suppress TNF-alpha production by LPS activated peritoneal cells. This control requires expression of MHC class II molecules, is independent of the inhibitory cytokines IL-10 or secreted TGF-beta and appears as cell contact dependent. Moreover, in this assay, regulatory activity is not abrogated when saturating amounts of IL-2 are added to the cultures. The physiological relevance of these in vitro assays is currently being tested in vivo.Immunology of Autoimmune Reactions, Max-Delbruck-Center for Molecular Medicine (MDC), Berlin, Berlin, Germany; 2 Laboratory of Neuroimmunology, IRCCS Santa Lucia, Rome, Rome, Italy.Objective: The identification of naive and memory subsets of T cells has been fundamental for the current understanding of the immune system. It is now generally accepted that regulatory CD25+CD4+ T cells are a central element of peripheral tolerance. Little is known, however, about phenotypic and functional characteristics of these cells with regard to memory. Aim of this study was therefore to identify regulatory memory cell subsets and to determine their markers and specific functions. Methods: T cells were purified by FACS/MACS-sorting. Phenotypic characterization (naive vs. memory, effector vs. suppressor) was carried out by flow cytometry, expression-levels of transcription factor foxp3 was determined by real time RT-PCR. Functional studies included in vitro proliferation and cell migration assays. As experimental in vivo systems congenic (SJL) and TCR-tg EAE models (TG4) were used. Induction of memory phenotype was demonstrated in vivo by adoptive transfer experiments of CFDA labelled cells. Results: In this study we show that the chemokine receptor CCR6 is expressed on a distinct subset of mouse Treg cells. Similar to their CD25counterparts, CCR6+ Treg cells exhibit markers of activation, memory and expansion that are indicative for an effector-memory function. They are memory-like cells, generated in vivo from CCR6-CD25+ T cells after the encounter of antigen. As conventional CD25-effector-memory T cells, they have a high turn-over rate and, in contrast to CCR6-Treg cells, they respond rapidly to restimulation in vitro with up-regulation of IL-10. CCR6+ Treg cells are enriched in the peripheral blood and accumulate in the CNS after induction of EAE. Importantly, these cells are also present in humans. Here the expression of CCR6 fully cosegregates with CD45RO, an established marker of human memory T cells. Conclusions: The subset of CCR6+ Treg cells seems to represent a population of dregulatory effector-memoryT T cells (T REM ), destined to control potentially destructive immune responses directly in inflamed tissues. T REM cells therefore seem to be the natural counter-players of effector-memory T cells (T EM ), involved primarily in front-linesuppression. Here we show that certain small molecules are able to reverse this process. We had recently identified a number of organic compounds that accelerate ligand exchange reactions of HLA-DR molecules in a similar fashion as the natural catalyst HLA-DM. In contrast to HLA-DM, however, these compounds are effective even at neutral pH and can therefore mediate ligand-exchange directly on the surface of living cells. Aim of this study was to identify the molecular mechanism by which small molecules mediate ligand exchange and whether these compounds can exhibit allele specificity. Methods: The experiments were carried out with soluble HLA.DR molecules expressed in insect cells. The kinetics of binding and release of peptide ligands was determined by ELISA experiments. The effect was validated in a cellular in vitro T cell system by using HLA-DR transfected fibroblasts as APC and by a flow cytometry based peptide binding assay. Importantly, some of these compounds were active only on a subset of HLA DR molecules. While they catalyse efficiently the loading of peptide ligands onto allelic variants such as HLA DR1, they completely failed to accelerate peptide binding to HLA DR2. Conclusions: Small molecular compounds can influence the ligand composition on the surface of antigen-presenting cells in an allele-specific manner. While they represent a novel class of molecular tools which can be used in therapeutic settings to facilitate antigen loading they might also represent environmental risk factors for allergy and autoimmune diseases. In this respect the strict allelespecificity of these compounds could even have an influence on the apparent linkage between incidence of certain diseases and HLA DR allele. Objective: It has been postulated that the bulk of B cell receptor (BCR) editing detected in peripheral B cell populations in both human and murine systems results from a tolerogenic process that occurs earlier in ontogeny, during B cell maturation. This possibility is substantiated by studies demonstrating that bone marrow B cells cultured ex vivo are capable of editing their BCRs when faced with self antigen. The purpose of the current study is to determine whether immature B cells isolated directly from murine bone marrow display evidence of BCR light chain editing.Methods: Hybridoma studies of IgH56R mice (with the anti-DNA 56R V H DJ H transgene inserted in the IgH locus upstream of C A ) have suggested that IgK editing is reflected in two ways: by preferential rearrangements to the Jk4 and Jk5 gene segments and also by skewed distributions of Vk gene usage. The hybridoma approach, however, is unable to distinguish between events early and late in B cell ontogeny because only mature splenocytes are captured via cell fusion. We have looked at IgK gene usage in a strictly defined immature B cell population from the bone marrow of IgH56R mice. B220 + CD43-IgD-cells further phenotyped as IgMa + IgMb-(expressing the IgH transgene) or IgMa-IgMb + (expressing an endogenous IgH) were single cell sorted into 96well plates. RT-PCR amplification and sequencing of expressed IgK genes were performed and identities of Jk segments and Vk gene families were assigned using the NCBI IgBlast website (www.ncbi.nlm.nih.gov/igblast).Results: Evidence of in vivo receptor editing in IgH56R bone marrow is revealed by comparing IgK gene expression in IgMa B cells (n = 143) to the IgMb control population (n = 184). Furthermore, the two subsets of immature B cells have significantly different predilections for Vk family gene usage (P = 0.0007). In particular, expression of anti-DNA generating Vk1 and Vk4 genes is substantially reduced in the IgH56R expressing cells versus controls (P = 0.017 and 0.006, respectively).Summary: This description of IgK gene expression in primary B cells from the bone marrow of IgH56R transgenic mice demonstrates that receptor editing begins as early as the immature B cell stage of development. Nonetheless, comparison of our observations to other sequence analyses of 56R B cells from a variety of sources clearly indicates that selective forces continue shaping the maturing B cell repertoire beyond this stage of development and probably well after their migration from the murine bone marrow to peripheral lymphoid tissues. Introduction: IgG immune complex (IC) induced inflammatory reactions can be triggered by two pathways; the complement system (especially C5a) or the cellular receptors for IgG, FcgR. In mice, IC can interact with either of three cellular receptors, two of which FcgRI and FcgRIII lead to cellular activation, while FcgRIIB is an inhibitory receptor. A hallmark of the immune response to IC is its ability to modulate the balance between these receptors. Recent reports identified C5a dependent inverse regulation of FcgRIII versus FcgRIIB on lung macrophages in the mouse model of acute alveolitis. Objectives: This study was carried out to analyze the underlying cellular and molecular mechanism of their cooperation in immuneregulation of IC lung pathology. Methods: The methods used in the study include adaptive cell transfer experiments in which alveolar macrophages (AM) depleted C5aR and FcgR deficient mice were reconstituted with ex-vivo modified AM from CD45.1 congenic mice. C5a:C5aR-dependent modulation of FcgR expression and its functional consequence was analyzed in presence of specific pathway inhibitors and quantified by IC injury, chemotaxis, TaqMan RT-PCR and FACS analysis. Results and Conclusion: Here we analyzed a novel regulatory C5aR-FcgR cross-talk on AM as the dominant event in the lung Arthus reaction, the classical animal model of immune complex (IC) disease. Strikingly, initial contact between IC and AM results in cellular regulation leading to plasma complementindependent C5a production; selective Ga i2 -dependent C5aR signaling; and C5aR-G i -mediated FcgR alterations towards FcgRIII, the main inducer of TNFa and CXCR2 ligand production. Distinct inhibitors of this refined inflammatory cascade are each effective in disease prevention, thus indicating cellular components of the C5aR-FcgR-axis, like Ga i2 , as potential new therapeutic targets in the treatment of inflammation and autoimmune diseases. Remarkably, our findings showed that macrophages function as an alternative source of C5a. These data also highlight the importance of local C5a-induced cellular mechanisms in disease pathogenesis.Supported by MD PhD Program, MHH and Deutsche Forschungsgemeinschaft (GE892/8-1).Su1.100. T Cells and Autoimmunity: Immunoregulation by CD40 Expressed on CD4 + T Lymphocytes. CD40 can stimulate autoreactive B cells directly, resulting in autoantibody production. However, recently published data indicate that autoaggressive CD4 + T lymphocytes also express CD40, including T cells from mice with collagen induced arthritis (CIA). Our lab has extensively studied signaling by CD40 in B cells and these reports prompted us to produce mouse (2B4.11) and human (Jurkat) T cell lines expressing transfected human (h)CD40. The objective of this study was to compare CD40 signaling in B vs.T cells, as well as characterize CD40 as a possible costimulatory molecule on T lymphocytes. As in B cells, CD40 in T cells binds to cytoplasmic adaptor proteins called TNF-R associated factors (TRAFs), including TRAF2 and TRAF3. However, CD40 mediated TRAF degradation is less efficient in T cells compared to their B cell counterparts. CD40 stimulation in T cells leads to JNK and NFnB activation and TNF-a production. Of particular interest is the ability of CD40 to act as a costimulatory molecule for T cell receptor signals, just as it does for the B cell receptor. Stimulating hCD40 transfected 2B4.11 T cells, or T cells expressing CD40 from mice with CIA, with anti-CD3, anti-CD28, or anti-CD40 alone leads to minimal secretion of IL-2 or IFN-g. However, stimulating via anti-CD3 in conjunction with anti-CD28 and/or anti-CD40 results in significantly increased secretion of these cytokines. This increase in cytokine production is paralleled by increased NFnB activation via both classical (NFnB1) and alternate (NFnB2) pathways, as well as increased activation of JNK. These findings are particularly exciting and suggest that T cell specific inhibition of CD40 signaling could be a means for blocking the autoimmune response without altering general immune function. Our lab has demonstrated that B-cell blasts, activated by LPS, anti-Ig or CD40L and transfected with a retrovirus encoding an IgG-peptide fusion protein are tolerogenic in both normal and primed recipients. Success has been achieved with multiple antigens in different mouse strains and in rats, and in preclinical models for MS, uveitis and diabetes. We also have demonstrated that class II MHC on the presenting B cells is necessary for tolerance and that the Ig carrier enhanced the degree and duration of tolerance. We have now adapted this system to a murine model for hemophilia A. Approximately 25% of hemophilia A patients make inhibitory antibodies that block clotting and therefore reverse the potential therapeutic benefit of factor VIII delivery. We know that most of these inhibitors are directed at epitopes in the C2 and A2 domains of factor VIII. Thus, for clinical application in hemophilia, we inserted residues S2173-Y2332 of the factor VIII C2 domain and S373-R740 of the fVIII A2 domain onto the IgG heavy chain backbone, respectively, to induce tolerance in hemophilia A mice. Specific tolerance to each domain was induced by this protocol. Importantly, a combination of A2-IgG and C2-IgG expressing B cells induced tolerance to the full length fVIII molecule, a result which supports the dominance of these domains in the immune response to fVIII. Tolerance was manifested in terms of ELISA titers, T-cell proliferation and especially Bethesda Unit titers (95% reduction). Importantly, similar results were obtained even when treatment was initiated after mice had preformed anti-fVIII titers, indicating its potential for the treatment of patients with binhibitorQ titers. Recent data suggest that CD25+ T regulatory cells are needed for in vivo tolerance induction with transfected B cells. These studies hold promise for a future clinical trial in hemophilia A patients. Viruses have been proposed as a triggering environmental event and some evidences have been reported, i.e. type I IFNs exist in the pancreata of diabetic patients and transgenic mice expressing these molecules in beta cells develop diabetes. Our group has generated the first NOD mice expressing a type I IFN (RIP-IFNbeta) in the islets, a new model of autoimmune diabetes. These mice develop accelerated diabetes at 3 weeks of age (equivalent to childhood in humans), with a cumulative incidence reaching 60% at 30 weeks age. This earlyonset diabetes is characterized by selective destruction of beta cells, MHC class I hyperexpression in the islets, severe insulitis, and a high number of Natural Killer cells in the pancreas. NK cells are innate immune cells that control certain virus infections and tumors, and have been recently associated to destructive forms of pancreatic islet autoimmunity in NOD mice. A significant increase of NK cells and NKT cells has been observed in the insulitis of the NOD RIP-HuIFNbeta and NOR RIP-HuIFNbeta transgenic mice at the early onset of diabetes when compared to healthy subjects. This high amount of pancreatic NK cells has not been found in transgenic mice or NOD wild type developing diabetes after 12 weeks of age. The percentage of NK cells in spleen and pancreatic regional lymph nodes is not altered in diabetic animals (early or late) when compared to healthy animals. This subset of NK cells is not maintained during the disease, decreasing quickly after 24 hours of the clinical onset. Transgenic mice NOD-Scid RIP-IFNbeta, unable to produce mature T and B lymphocytes although they have unaffected NK subset, do not develop diabetes thus suggesting the need of interaction between NK cells and other lymphocyte subsets (T, B) for autoimmunity. Microarray experiments demonstrate a correlation of the NK cell subset in early onset diabetes to the islet expression of adhesion molecules, costimulatory molecules (CD86), cytokines (IFNgamma, IL6) and NK attractant chemokines: CCL2 (MCP-1), CCL3 (MIP-1alpha), CCL5 (RANTES), CXCL10 (IP10) and XCL1 (lymphotactin). Since type I IFNs production takes place in cells infected mainly by viruses, our transgenic model could be an interesting tool for studying how the production of IFNbeta causes cellular stress, generates danger signals, inflammation and eventually NK cellmediated autoimmunity. The characterization of the role of NK cells at early onset of T1D may help us to understand and to prevent this autoimmune disease. Dendritic cells (DCs) are potent antigen presenting cells capable of antigen uptake and presentation. In the immature developmental stage DCs are thought to induce T-cell anergy. In the steady state, meaning in the absence of acute infection and inflammation, DCs deliver the antigen to T cells without of essential co-stimulatory molecules inducing T cell tolerance. The key role of DCs in the induction of immunity to infectious agents, malignancy and transplanted allografts, and in the maintenance of T cell tolerance in the periphery has prompted the interest in their use as immunotherapeutic agents.OBJECTIVE: In this study we examine whether bacterial lipopeptide (BLP) may induce phenotypic and functional changes associated with DCs maturation through Toll-Like Receptor 2 (TLR2). METHODS: DCs were generated from bone marrow precursors (BMDCs) by using granulocyte-macrophage colony stimulating factor (GM-CSF). On the days 3 and 5 appropriated cytokines were added. CD11c, MHC-II, CD80 and CD86 surface molecules expression was determined using murine monoclonal antibodies conjugated to phycoerithrin (PE) and isocthiocyanate (FITC) by flow cytometry. The capacity of BMDCs to endocytose antigen was determined by flow cytometry using FITC-conjugated dextran. RESULTS: We demonstrated that BMDCs generated in vitro by using GM-CSF and BLP for two days show a high MHC-II expression and an increased yield of differentiated DCs. These phenotypic features of DCs correspond to a semi-mature (sm-DCs) developmental stage. In contrast, bone-marrow precursor cultured for five days under BLP stimulus or alternative standard protocols using TNF or LPS for over eight days yield the same number of differentiated DCs and a mature phenotype demonstrated by functional assays. CONCLUSION: This protocol has shown to be more rapid and efficient to generate sm-DCs with tolerogenic characteristic and mature DCs that will be use in immunological therapy.Financed Background and Objectives. Mucosal administration of antigen is an established method to induce immunologic tolerance and mucosal administration of auto and alloantigens is effective in treatment of animal models of autoimmunity, inflammation, and transplantation. Parenteral administration of anti-CD3 is efficacious in animal models of autoimmunity and in humans anti-CD3 is an approved therapy for transplant rejection and positive results have been reported in patients with new onset type 1 diabetes treated with parenteral anti-CD3. We investigated the effect of mucosally administered anti-CD3 in animal models of autoimmunity and transplantation. We orally or nasally administered anti-CD3 or FabT2 fragments of anti-CD3 (or appropriate isotype control antibody) in doses ranging from 0.5ug to 500ug. The PLP specific immune response of anti-CD3 fed animals immunized with PLP demonstrated a decreased proliferative response, reduced IL-2 secretion and increased secretion of IL-10, IL-4 and TGF-b. Oral anti-CD3 was associated with an increase in the numbers of CD4+ TGF-b latency associated peptide (CD4+LAP+) in the mesenteric lymph node. CD4+ LAP+ cells suppressed proliferation of CD4+CD25-LAP-T cells in vitro and adoptive transfer of CD3+LAP+ cells suppressed EAE in a TGF-b dependent fashion. No modulation of CD3 on the surface of CD4+ T cells occured after oral anti-CD3. Similar results were obtained in MOG induced chronic EAE in the NOD mouse. Pathologically there were less CD4+ cells and macrophages in the spinal cord. In the NOD model of diabetes, oral anti-CD3 given in the neonatal period suppressed the incidence of diabetes. In streptozocin induced diabetes oral anti-CD3 FabT2 suppressed diabetes in association with CD4+LAP+ cells whose suppressive function increased after oral anti-CD3. In vivo neutralization of TGF-b reversed the suppressive effect. In the DBA2 model of collagen induced arthritis, nasal anti-CD3 FabT2 suppressed the incidence of arthritis, was associated with decreased levels of TNF in the joints, and was more effective that mucosally administered collagen. In allogeneic cardiac transplantation (Balb/c into C57BL/6), oral anti-CD3 was given on day -5 and continued until day +10 post transplantation. Cardiac transplants in mice receiving oral anti-CD3 survived an average of 16.2 days vs. 8.4 days for controls (P = 0.0004). Oral anti-CD3 induces regulatory T cells characterized by surface LAP that function in a TGF-b dependent fashion. These results identify a novel and physiologic mechanism to induce regulatory T cells that is clinically applicable to a variety of immune mediated disorders. HIT is a serious complication of heparin therapy caused by antibodies (Ab) to complexes between high molecular weight heparin and an endogenous protein, Platelet Factor 4 (PF4), leading to limb and/or life-threatening thrombosis in~50% of affected patients. We are interested in understanding the basis of an important clinical observation: HIT auto-Ab form in most heparinized patients, but only a small percentage of these develop thrombocytopenia and/or thrombosis. In the present work we show by gel filtration that PF4 and heparin form antigenic ULC over a very narrow molar ratio (~1:1) of reactants. ULC are stable and visible by electron microscopy, but can be dissociated into smaller complexes (SC) upon addition of heparin. Mutation studies show that formation of ULC depends on the capacity of PF4 to form tetramers, which then assemble into multimolecular lattices on a heparin scaffold. Additional of PF4 to human or mouse platelets and other vascular cells leads to the self-assembly of antigenic complexes presumably nucleated by membrane glycosaminoglycans (GAG). PF4 evokes binding of HIT-like monoclonal Ab to cells in a dose-dependent manner over a narrow range of concentrations and induces FcgRIIAdependent platelet activation analogous to ULC. We conclude that the capacity of PF4 to form ULC composed of multiple PF4 tetramers arrayed in a lattice with several molecules of heparin and/or GAG may play a fundamental role in autoAb formation. The capacity of ULC to bind multiple Ab and cross-link/trigger FcgRIIA promotes platelet activation and explains the propensity for thrombosis. We are currently testing the hypothesis that the patients with high steady-state level of surface PF4 based on genotype and acquired through platelet activation are those most likely to generate autoAb, form ULC and develop HIT. Objective: To compare and optimize freezing protocols for PBMCs taken from whole blood for detection of autorective T cells.Background: To detect the effects of potentially immunomodulatory drugs on autoreactive T cells, clinical trials performed by the Immune Tolerance Network employ assays that quantify T cell responses to autoantigens. Centralized assay facilities are used to reduce the problem of inter-site variability. However, some intersite variability can occur during the preparation of PBMC samples at various geographic locations. We have therefore examined the effects of our freezing and thawing procedures to minimize inter-site variability of the results of T cell assays.Methods: PBMCs from healthy donors and MS patients were isolated by ficoll separation. Parts of the samples were exposed to different doses of tetanus toxoid, myelin antigens, PHA or no antigen, while the other part was frozen for 3 weeks using various freezing media and temperatures prior to stimulation in T cell assays. Proliferation ([ 3 H]-Thymidine incorporation), cytokine production (ELISA, flow cytometry (FACS) and elispot) and cell death/apoptosis (FACS) were assayed in parallel in both fresh and frozen samples. Subpopulations of PBMCs were phenotyped by FACS analysis.Results: Cell viability and recovery decreased after a cycle of freezing/thawing (2 to 10% and 20 to 60%, respectively). The viability as well as PBMC response to antigens were significantly improved when human AB serum (+10% DMSO) was used to freeze the cells. The viability of PBMCs using fetal bovine serum (+10% DMSO) resulted in a fair viability (91%) but high background for proliferation and cytokine production. Using cold (48C) freezing media decreased both the viability (by 4%) and the response of T cells to antigens (by 20%). PBMCs were better preserved (numerically and functionally) and retained a greater response to antigens when the temperature of freezing medium used was kept at 258C than 48. Using these optimal conditions we did not find significant differences in the cell population percentage between the fresh and frozen regarding CD3, CD4, CD8, CD14, CD19, activated or memory T cells.Conclusions: We have found as expected that freezing and thawing of PBMCs decrease cell viability (2 to 10%) and T cell response (50%). This decrease in count and viability did not disproportionately affect a specific cell population among those examined. The best combination of freezing conditions was obtained using human AB serum+10% DMSO at 258C. Thus, we have identified procedures optimal for PBMC viability and T cell responses to PHA and a panel of nominal and self-antigens. This protocol has been adopted as standard operating procedure for all studies conducted by the ITN and therefore expands the ITNTs capability to evaluate mechanisms of disease and treatment response in multicenter trials. MBL is a member of the collectin family with structural similarities to the lung collectins, SPA and SPD. Like C1q, MBL is a circulating serum protein that is sequestered to sites of inflammation and infection. Limited reports of patients deficient in MBL have raised the possibility that lack of MBL, in a manner similar to C1q deficiency, might be associated with autoimmunity. Cells dying by apoptosis are an important target for the autoantibodies that develop in systemic lupus and failure of removal of dying cells has been implicated in development of autoimmunity in C1q deficiency. Here we show that MBL, like the other collectins and C1q, is able to bind apoptotic cells in vitro. Using mice deficient in both mouse MBL genes, MBLA and MBLC, we demonstrate that MBL null animals show a 50% defect in their ability to clear apoptotic cells, confirming a role for MBL in clearance of apoptotic cells in vivo.Analysis of MBL null animals demonstrated expanded B1 cells but no spontaneous activation of antigen presenting cells. Importantly, despite demonstrating a defect in apoptotic cell clearance comparable to that seen in the C1qA-/-animals, MBL null animals did not develop spontaneous autoimmunity, lymphoproliferation or germinal centre expansion. These data demonstrate an important in vivo role for MBL in clearance of dying cells and add the MBL null animals to the short list of animals with demonstrable in vivo apoptotic cell clearance defects. Furthermore, it demonstrates that failure of apoptotic cell clearance can be dissociated from autoimmunity indicating that other factors must be required for autoantibody generation and end organ damage. Autoimmune thyroid disease (AITD) is a term that includes various clinical entities, among them HashimotoTs thyroiditis (HT) and GravesT disease (GD). Lymphocytic infiltration by T-, Blymphocytes and DC and FDC that is often organized as functional lymphoid follicles with germinal centers is an almost constant feature. In order to investigate specific homing of recent thymic emigrants (RTE) to the thyroid and the distribution of recent RTEs in the different subsets of PBLs in patients with AITD, TCR excision circles (TRECs) have been measured in CD3+ intrathyroidal lymphocytes (ITL) and in peripheral blood (PBMC) of the same patients. TRECS were measured by real-time PCR in CD3+ lymphocytes obtained by cell sorting from dispersed cell preparations of thyroid glands (n = 10) and from peripheral blood from (n = 15) patients and (n = 12) healthy controls. The comparison of TREC levels ITLs and PBMCs in each individual showed two patterns: in one half of patients TRECs in ITL were higher than in PBMCs whereas in the other half was higher in PBMCs. On the other hand TREC content in PBMC from AITD patients was not significantly different from controls (1.799 F 2.62/10 4 cells vs. 3.212 F 3.06/10 4 cells, P = 0.902, t test). To assess the influence of T cell proliferation on TREC levels, we measured telomere length in B-and Tlymphocytes in groups of 10 patients with AITDs (ITL and PBMC) and controls (PBMC). In AITD patients, the relative telomere length (RTL) in ITL was significantly lower than in PBMC ( Several autoimmune diseases such as psoriasis, multiple sclerosis and CrohnTs disease, are associated with an imbalance between Th1/Th2 towards Th1 cells. This creates a state of chronic inflammation with predominance of Th1 cytokines such as IFNg , TNF-a and IL-2. In contrast, the Th2 cytokines like IL-4 and IL-10 have shown beneficial effects and manipulation of Th1/Th2 balance is part of the current strategy developed against Th1-mediated immune diseases. 1a,25-dihydroxyvitamin D 3 (calcitriol) has been shown to exert several immunomodulatory functions on T cells and antigen-presenting cells (APC) both in vitro and in vivo. Our goal was to evaluate the immunomodulatory effects of a new vitamin D analog, QW1624F2-25SO2-1, for the therapeutic treatment of autoimmune diseases. We also examined, using ELISA, the effect of our compound on Th1/Th2 cytokines in human peripheral blood mononuclear cells (PBMC) and on cytokines secreted by macrophages. The use of vitamin D analogs is currently limited by the induction of hypercalcemia; therefore, the effect of QW1624F2-25SO2-1 on serum calcium level was evaluated in mice. Results: Transcriptional analysis in human PBMC revealed that calcitriol is a strong inducer of the VDRresponsive gene CYP24 which indicates these cells are responsive to VDR-mediated gene transcription. In a macrophage cell line, very low concentrations of QW1624F2-25SO2-1 significantly induced CYP24 expression. Interestingly, the ability of this analog to bind to VDR was weaker than calcitriol. Calcitriol has been shown to repress IFN-g, GM-CSF, IL-2 in T cells and IL-12 in APC, an effect that is dependent on the presence of VDR. Our results revealed that QW1624F2-25SO2-1 significantly decreased pro-inflammatory Th1 cytokine production (IFN-g, TNF-a and IL-2) and increased the production of the Th2 cytokine IL-4 in human PBMC. This analog also affected macrophage functions by inducing the secretion of IL-10 in LPS-stimulated U937 cells. In vivo, this analog did not induce hypercalcemia even at the highest dose of 40 Ag/kg body weight. Conclusion: These results demonstrate that QW1624F2-25SO2-1 is a low-calcemic analog and a strong inducer of vitamin D-dependent transcription. This novel compound modulates Th1/ Th2 balance in favor of the Th2 and macrophages function suggesting its potential for the treatment of Th1-mediated autoimmune diseases.Su1.110. SP-A May Be a Candidate of bQiQ Molecule Triggers Some Autoimmune Diseases.of SLE. We found that hCDR1 treatment of BALB/c mice resulted in a marked elevation of expression of TGFh in vivo, and in TGFhinduced suppression of 16/6Id-stimulated T-cell proliferation exvivo. In addition, we provide evidence that one possible mechanism underlying the hCDR1 and TGFh-induced inhibition of T-cell proliferation is by down-regulating the expression, and therefore, the functions, of a pair of key cell adhesion receptors, LFA-1 (aLh2) and CD44, which operate as accessory molecules in mediating antigen presenting cell (APC)-T-cell interactions. Indeed, T cells of mice treated with hCDR1 showed a TGFh-induced suppression of adhesion to the LFA-1 and CD44 ligands, hyaluronic acid and ICAM-1, respectively, induced by SDF-1a (CXCL12) and PMA. The latter suppression is through the inhibition of ERK phosphorylation. Thus, the down-regulation of SLE by hCDR1 treatment may result from the influence of the up-regulated TGFh on the expression and function of T-cell adhesion receptors, and consequently, T-cell stimulation, adhesion, and proliferation.hCDR1 ( Rationale: Numerous studies have been performed in vitro and in various animal models to modulate the interaction of DC and T cells by Fas (CD95/Apo-1) signaling to delete activated T cells via induction of activation-induced cell death (AICD). However, similar studies with primary human cells have not been performed. Recently, we could demonstrate that Fas Ligand (FasL/CD95L)expressing bKiller-DCQ can be generated from human monocytederived mature DC using adenoviral gene transfer. To evaluate, whether these FasL-expressing DC (DC-FasL) could eliminate human CD8+ T in vitro, coculture experiments were performed.Methods: Human CD8+ T cells and a CTL clone specific to the HLA-A2 binding Melan-A26-35 peptide were activated in a first mixed lymphocyte reaction (MLR) with mature DC pulsed with Melan-A peptide. Activated T cells were rescued and a second MLR was established with either DC-FasL, EGFP-transduced control DC (DC-EGFP) or untreated DC loaded with Melan-A-or control peptide at different ratios. As a read-out system proliferation (thymidine incorporation) and apoptosis (Annexin V/PI staining) of T cells were determined.Results: FACS-analysis of PKH26 labeled DC revealed that FasL-transduced DC but not DC or DC-EGFP loaded with Melan-A peptide were protected from cytotoxicity mediated by Melan-A specific CD8+ T cells. In addition, no proliferation could be observed in Melan-A specific CD8+ T cells cocultured with DC, DC-EGFP or DC-FasL loaded with control peptide. In contrast, proliferation of activated Melan-A specific CD8+ T cells was markedly reduced in cocultures with Melan-A peptide-loaded DC-FasL, whereas a strong secondary proliferative T cell response could be observed in cocultures with DC-EGFP or DC. Inhibition of T cell proliferation was directly related to the numbers of DC-FasL present in cocultures, and at a ratio of 1:1, T cell proliferation was completely inhibited by DC-FasL, which was due to induction of apoptosis in the majority of Melan-A specific CD8+ T cells (approximately 70%). Spontaneous apoptosis detected in CD8+ T cells cocultured with Melan-A peptide-loaded DC or DC-EGFP was low (approximately 25%).Conclusion: The present results demonstrate for the first time that human DC-FasL were protected from CTL-mediated cytotoxicity (counter attack). In addition, Melan-A specific CD8+ T cells were efficiently eliminated by Melan-A peptide-loaded DC-FasL, supporting the concept to apply FasL-expressing bKiller-DCQ as a novel strategy for the treatment of T cell dependent autoimmune disease. Gai2 À/À mice are deficient in the formation of certain B and T cell subsets and are susceptible to immune dysregulation, notably developing inflammatory bowel disease (IBD). A key issue is the identity of the Gi-coupled receptors mediating this Gai2 requirement for lymphocyte development. Here, we test the prediction that CB2, the Gai2-coupled peripheral endocannabinoid receptor, is one such receptor. B and T cell subsets, isolated from tissues of the peripheral, mucosal, and serosal lymphoid compartments of CB2 null and sufficient mice were quantified by flow cytometry. Mice bearing the CB2 null phenotype had profound deficiencies in both B and T cell subsets, particularly splenic marginal zone (MZ) and peritoneal B1a cells, as well as splenic memory T cells and intestinal NK and NKT cells. CB2 is required for the formation of many immunoregulatory B and T cell subsets. These findings phenocopy and extend the developmental disorder associated with Gai2 À/À and suggest that the endocannabinoid system is required for the formation of T and B cell subsets involved in immune homeostasis. Microflora in gut of IBD patients becomes aberrant, with normal microflora decreased, harmful and potential harmful bacteria increased. As have been reported, there has close relationship between gut aberrant microflora and mucosal immune function disorder, and modification of intestinal microflora may have therapeutic effect on IBD. In the experiment, we used dextran sulfate sodium(DSS)-induced colitis in mice which has same pathological property as human IBD, and fed mice with Bifidobacterium before inducing colitis, to study the therapeutic effect of supplement with bifidobacterium on pathogenesis of colitis, and explore the possible mechanism. Inflammatory Bowel Diseases METHODS: Mice were randomly divided into four groups: Control,DSS,SASP,and Bifidobacterium(Bif in short). Mice of groups DSS,SASP,and Bif were fed with 5% DSS(w/v) solution for 7 days to induce colitis, mice of Control just drink distilled solution, and disease activity index (DAI) was calculated every day. Mice of SASP were fed with SASP every day during inducing colitis, and mice of Bif were given Bifidobacterium by oral gavage from 7 days before the experiment to the end of experiment. The expression of TNF-a,NF-nB P65,Fas and MPO in inflamed colon of each group mice was measured at the end of experiment.RESULTS: Mice of group SASP,Bif showed lower DAI than those of group DSS since the forth day of experiment. There were lower expression of TNF-aand MPO in murine inflammatory colon, lower NF-nB P65 appearance in nuclei of inflammatory cells, lower Fas expression in colonic epithelia of group SASP,Bif compared with group DSS at the end of experiment.CONCLUSION: Treatment with bifidobacterium has beneficial effect on murine experimental colitis, the mechanism may be involved in such respect: Bifidobacterium inhibits proinflammatory cytokine secretion and NF-nB activation in inflammatory cells, limits colonic inflammation, downregulates Fas expression in colonic epithelia of murine inflamed colon, alleviates inflammatory damage of colonic epithelia and protects the integrity of intestinal mucosal barrier.Key words: probiotic experimental colitis Dextran sulfate sodium inflammation Su1.115. Anti-Murine TNF-alpha Reverses TNBS Colitis in Mice but Not Oxazolone Colitis: Potential Role of Apoptosis Induction. However, administration of infliximab (a chimeric anti-TNF mAb) has shown beneficial effect in clinical trials in CD but is less effective in UC. Aim: 1) to investigate the effect of anti-TNF on trinitrobenzenesulphonate (TNBS) and oxazolone (Oxa) colitis in mice as models for CD and UC respectively; 2) to study the apoptosis-inducing effect of anti-murine TNF both in vitro and in vivo in mice. Method: 1) Colitis was induced by rectal administration of 1mg TNBS or Oxa in 50% ethanol after 2 pre-sensitizations via the skin. Anti-murine TNF-alpha was given intraperitoneally (i.p.) 2) Thioglycollate-elicited peritoneal macrophages were treated in vitro with LPS and anti-murine TNF-alpha or non-specific control antibody. Annexin V & propidium iodide and 7AAD were used to study apoptosis on the cell membrane and DNA level respectively.3) For in vivo analysis of anti-TNF effects on macrophages, antimurine TNF-alpha or control antibody was administrated to SCID mice. Peritoneal macrophages were recovered and apoptotis was analyzed as described above. Results: 1) In the TNBS colitis model, mice treated with anti-TNF recovered more rapidly compared to the control treated mice. Histological analysis revealed less severe signs of colitis; on the contrary, no beneficial effect of anti-TNF was found in the Oxa colitis mice. 2) Apoptosis was induced by anti-murine-TNF in peritoneal macrophages. In vitro, apoptotic cells in the presence of anti-TNF amount to~40% compared to~20% in the control cultures. In vivo,~30% less macrophages could be harvested from the anti-TNF treated group. A higher percentage of peritoneal macrophages were apoptotic (~50%) compared to the control group (~25%). These results correlate with difference in efficacy of antihuamn TNF treatment in UC and CD respectively. The apoptosis inducing-effect of anti-TNF could be demonstrated in peritoneal macrophages. The data suggest a different involvement of TNF expressing cells in the pathogenesis of both disease models. Introduction: Increasing evidence indicates that granulocyteapheresis (GCAP) is effective and safe therapeutical strategy in treatment of the ulcerative colitis (UC). However the ideal treatment scheme is not established yet.Objective: This study was carried out to evaluate the efficacy and safety of 5 as compared to 10 GCAP treatments in patients with moderate active steroid dependent UC.Matherials and methods: In this prospective multicenter randomized clinical trial 20 patients were randomized to 5 or 10 GCAP treatments (1 weekly). Each treatment consisted in a 1hour apheresis session with AdacolumnR at 30 ml/h with 1,8 l of blood being processed. Secondary variables included CAI at all weeks, quality of life questionnaires (IBDQ and EuroQoL), endoscopic activity index (EAI) at week 17, as well as steroid consumption and analytical parameters.Inclusion criteria were: active UC (CAI between 6 and 12), EAI N4, total colon affected length N25 cm and steroid dependency defined as at least one unsuccessful attempt to taper down steroids plus use of N400 mg of prednisone within 4 weeks prior to the study start. Clinical remission was defined as CAI V4 and clinical response as a drop in CAI z3.Since some patients are still in the follow-up, efficacy for each treatment regimen, as well as other secondary variables analysis will be reported on completing the study.Results: 12 males and 8 females were included, being the mean age 41, 7 F 15, 2 years and mean disease duration 80, 7 months (6-528). Two patients had chronic active UC whereas mean number of flare ups during the year prior to the study entry was 2, 2 F 1 for remaining patients. All patients were previously treated with 5-ASA and steroids, 55% with immunosuppressants and 15% with cyclosporine. 9 patients were randomized to 5 GCAP sessions and 11 patients to 10 GCAP sessions. Five out of 11 patients were steroid free at week 11. Two not serious adverse events, and one community pneumonia were reported.Conclusions: Granulocyteapheresis is a safe and effective treatment for moderate active steroid dependent ulcerative colitis. In addition, it shows an important steroid sparing effect in a severely steroid dependant population, even allowing complete steroid withdrawal.Su1.117. Cytokine and Chemokine Transcript Profiles in Acute Pouchitis.A. Stallmach, 1 T. Giese, 2 C. Schmidt, 3 B. Ludwig, 3 S. Meuer. 2 1 Gastroenterology, Catholic Clinics Essen-Nord, Essen, Germany; 2 Immunology, University of Heidelberg, Heidelberg, Germany; 3 Internal Medicine II, Saarland University, Homburg, Germany.Background: After ileo-anal pouch anastomosis (IAP) 10-40% of patients with ulcerative colitis (UC) but only 5% of patients with polyposis coli (FAP) develop a pouchitis. Therefore we characterized cytokine and chemokine transcripts in inflamed and non-inflamed pouches in patients with UC and FAP.Methods: Mucosal biopsies were taken from 42 patients with IAP (UC (n = 37) or FAP (n = 5)). Patients with active ileal Crohńs disease (CD; n = 14), active UC (n = 8), specific colitis (infectious colitis, ischemic colitis; n = 15) and patients with non-inflammatory conditions (n = 13) served as controls. Expression of 30 proinflammatory gene transcripts were quantified using real-time PCR.Results: Compared to normal ileal mucosa from controls, biopsies from non-inflamed mucosa from IAP (UC and FAP) patients expressed elevated transcript levels for MRP-14, IL-8, IL-1h, IFN-g and MIP-2a. In UC-IAP patients MRP-14 (2,9-fold) , IL-8 (2,6-fold) and IL-1h (3,8-fold) transripts were elevated in their inflamed mucosa in comparison to their noninflamed mucosal biopsies. No differences were found concerning TNF-a, IP-10, IL-18 and ELC. Levels of TNF-a, IFN-g and IL-23 were elevated in inflamed CU pouch mucosa in comparison to specific colitis, suggesting a predominantly Th1 mediated inflammation. A good correlation between pouchitis activity (PDAI) and MMP-1 and MIP-2a transcripts was observed.Discussion: In acute pouchitis in UC patients after IAP anastomosis transcript levels of pro-inflammatory cytokines and chemokines of predominantly Th1 origin were found elevated, even if these data cannot completely explain the immunolgical etiology. Inflammatory Transcript Profiles Reflect Onset of Clinical Remission in Patients with Steroid Refractory CrohnTs Disease after Treatment with Cyclophosphamide or Infliximab. All probiotic treatment was discontinued when remissions occurred, (although we discovered later that probiotics needed to be continued long-term. However most eventually relapsed. The exceptions were four patients who sustained complete remissions for over 10 years. They also elected to continue to take prophylactic probiotics. Follow up colonoscopies and biopsies were normal. In one case, AP, the donor strain disappeared from feces following prophylactic ceftin therapy for a knee replacement. He remains well 12 years after he declined colectomy. She remains well and continues to take both E. coli (Mutaflor, Ardeypharm Co, Germany) and L. acidophilus. Her 20 year old brother was also treated 10 years later but he did not colonize. He is improved but unlike his sister, he has not sustained a complete remission. Other cases with intermediate results will be presented which suggest that this approach should be pursued with controlled studies and especially using continued prophylactic probiotics containing a non-pathogenic E. coli. Interleukin 21 (IL-21), a member of the common gamma-chain family of cytokines, is secreted by activated T cells and can have a diverse range of immunomodulatory effects dependent upon the particular context of the immune response. Interleukin 21 Receptor (IL-21R) is expressed on many immune system cell types including activated T cells. Rationale: In situ hybridization studies have shown that IL-21R is highly expressed in the lymphoid compartment in the gut, and the human IL-21R gene has been mapped to chromosome 16 within the CrohnTs Disease susceptibility region, suggesting that the IL-21 pathway may be involved in regulation of gut homeostasis. We examined the role of IL-21 in a T cell mediated model of intestinal and skin inflammation. Methods: CD45RB hi CD4 + naive T cells were transferred into severe combined immunodeficient (SCID) mice and housed under different caging conditions so that the mice developed colitis or colitis with skin lesions resembling psoriasis. Mice were treated with soluble murine IL-21RFc or an IgG2a control Antibody and assessed for development of disease. Purified CD45RB hi cells were also cultured with anti-CD3 and IL-21 or IL-21RFc to determine the effect on proliferation and cytokine secretion. Results: In culture, anti-CD3 stimulated CD45RB hi (naive) but not CD45RB lo (memory) CD4 + T cells proliferated in response to IL-21 and secreted increased levels of IL-2, IL-4, IL-10, IL-17, IL-18, IFN-g and TNF-a. Blockade of endogenous IL-21 with neutralizing soluble IL-21RFc resulted in decreased levels of cytokines in these cultures. In mice that developed skin inflammation, treatment with thrice-weekly IL-21RFc seven weeks after CD45RB hi cell transfer, resulted in reduced erythema, scaling and hair loss when compared to IgG2a-treated controls. Treatment of CD45RB hi recipient mice with 200 ug IL-21RFc, 3 times per week at the time of cell transfer, resulted in a significant reduction of clinical signs of colitis as measured by body weight loss and stool score when compared with control -treated mice. Macroscopic evaluation of colons from control treated CD45RB hi recipients showed severe thickening and swelling which was almost completely suppressed in mice treated with IL-21RFc. Microscopically, control-treated mice also exhibited a greater degree of epithelial hyperplasia and leukocyte infiltration in the lamina propria/submucosa when compared with IL21RFc-treated mice. Conclusions: Taken together these results suggest that IL-21 is a potent potential player in the inflammatory responses in this model and blockade of this pathway may be of therapeutic benefit in Th1 mediated diseases such as CrohnTs and psoriasis. Inducible costimulator (ICOS) is a CD28 homologue that is induced upon T cell activation. ICOS binds to its ligand ICOSL which is expressed on fibroblasts, endothelial cells, some epithelial cells and constitutively at low levels on resting B cells, on some macrophages and dendritic cells. ICOS ligation enhances T cell proliferation and the production of several cytokines such as IFNg, IL-4 and has a key role in IL-10 production. IL-10 plays an important role in the induction of regulatory T cells and suppression of autoimmunity. We have used ICOS deficient (À/À) mice to investigate the role of ICOS on 1) the induction and function of regulatory T cells, and 2) the function of pathogenic effector T cells using the colitis model developed by Powrie and colleagues. In this colitis model, the transfer of CD4 + CD25-CD45RBhigh T cells (Teff) from normal mice to C.B-17 SCID recipients leads to the development of a Th1-mediated inflammatory bowel disease similar to IBD in humans. Intestinal inflammation is the result of the development of a Th1 response driven by enteric bacteria. Colitis induced by transfer of Teff cells can be prevented by cotransferring cells contained within the CD4 + CD25 + CD45RBlow population (Treg) .Surprisingly, we found that ICOSÀ/À regulatory T cells protected mice from colitis, indicating that ICOS is not required for the induction of a functional regulatory T cell population. However, we found that wild type regulatory T cell could not protect from IBD induced by T effector cells lacking ICOS. Without ICOS the balance appears to be shifted in the favour of autopathogenic Th1 cells, such that these effector cells cannot be controlled by regulatory T cells, thereby resulting in more severe clinical disease. Thus, these studies suggest that ICOS is a critical regulator of the balance between regulatory T cells and effector T cells. Although CrohnTs disease (CD) is associated with low IL-4 production by T-bet-expressing Th1 cells in the lamina propria, surprisingly a higher expression of c-Maf in these cells was found as compared with control patients. The relevance of this finding was further evaluated in an animal model of CD induced by adoptive transfer of CD4 + CD62L+ T cells in RAG-deficient mice. In this Th1-mediated model, an increase of c-Maf-expressing T lymphocytes in the lamina propria over time was observed. Interestingly, adoptive transfer of c-Maf transgenic CD4 + CD62L + T cells in RAG-1-deficient mice resulted in an IL-4-dependent inability to induce colitis and suppressed colitis activity induced by wild-type CD4+CD62L+ T cells.In contrast, transfer of CD4+CD62L-T cells from c-Maf transgenic, but not wild-type mice, induced colitis and augmented a colitis induced by CD4 + CD62L+ T cells from wild-type mice in an IL-4-independent pathway, as determined by macroscopic, histologic, and endoscopic criteria. This was associated with an accumulation of CD4+ T-bet + CD25 + effector Th1 cells in the lamina propria of colitic mice. Although overexpression of c-Maf in naive T cells prevents Th1-mediated colitis, overexpression of c-Maf in memory T-bet+ Th1 cells regulates CD25 expression and augments such colitis. Targeting of c-Maf in memory T cells in CD appears to be an attractive target for therapeutic interventions.Su1.123. Dual Immune Suppressive Activity of 4AZA1378 Alleviates TNBS-Induced Colitis in Mice. Introduction: Elevated production of TNF-alpha and activated T cells play a central role in the pathogenesis of CrohnTs disease (CD). Recently, 4AZA1378 was identified as a phosphodiesterase-4 (PDE4) inhibitor (IC50; 31 nM), which explains its inhibitory effect on LPS-induced TNF-alpha production in vitro (IC50; 245 nM) as well as in vivo. PDE4 inhibition can however not account for the strong inhibitory effect in the Mixed Lymphocyte Reaction (MLR) assay (IC50; 4 nM) which suggests another target in this assay. The latter was confirmed by the inability of Rolipram (a specific PDE4 inhibitor) up to 50.000 nM to inhibit the MLR. Aim: To investigate the efficacy of 4AZA1378 in trinitrobenzenesulphonate (TNBS) induced colitis in mice, a model of CrohnTs disease. Methods: Colitis was induced by rectal administration of 1mg TNBS in 50% ethanol after 2 pre-sensitizations via the skin. 4AZA1378 (20 mg/kg) was given intraperitoneally daily. Results: Mice treated with 4AZA1378 had less severe signs of colitis and recovered more rapidly, as evidenced by more rapid weight recovery, and histologically by a reduction of inflammatory lesions, less edema, a reduction of goblet cells loss and reduced wall thickness. Cell infiltration, especially infiltration of neutrophils, as shown by myeloperoxidase activity, was reduced in 4AZA1378 treated animals. Experimental and clinical studies have indicated that cytokines play an essential role as mediators of inflammatory process associated with pancreatitis. One of the most important mediators of inflammation is interleukin-6 (IL-6). Interleukin-6 is a 22-30-kDa glycoprotein produced by many cell types and has a wide variety of biologic, differentiation, and growth-promoting effects in a variety of target cell types.The importance of IL-6 in the acute phase has been confirmed by the observation that it stimulates the synthesis of acute phase proteins, including C reactive protein (CRP), from hepatocytes in vitro and in vivo. IL-6 levels are raised in patients with acute pancreatitis (AP) and correlate with disease severity. Serum concentrations IL-6 of patients during the first 48 hours of hospitalization is a valuable marker permitting the differentiation of various types of pancreatitis (AP, CP-chronic pancreatitis, CEPchronic exacerbated pancreatitis).The immunhistochemical detection of IL-6 in pancreas during pancreatitis, to our knowledge, has not been previously described. In previous studies, the presence of IL-6 in human pancreatic cells had only been shown on biopsy material from diabetic patients or normal gland.AIMS & METHODS:The aim of the study was to identify immunohistochemically the localization of IL-6 and to determine IL-6 expression in CP and CEP. Samples of tissues of normal pancreas (n = 5) (obtained at autopsy) and CP (n = 14), CEP (n = 2) were verified histopathologically and then IL-6 was localized by immunohistochemical staining using the monoclonal anti-human IL-6 antibody (R&D Systems USA) and test LSAB2-HRP (DAKO,USA) to visualize IL-6/Ab complexes. RESULTS: We found only scare acinar cells staining positively for IL-6 in the normal human pancreas (À/+); islets cells did not show IL-6 immunoreactivity. In slices of the pancreas, derived from patients with CP and CEP, a much stronger immunohistochemical reaction (+ +; + + +; diffused and focal) was noticed as compared to controls. IL-6 was localized in exocrine and islet cells of the pancreas. The immunohistochemical reaction of ducts cells was also strong. Interestingly, this cytokine was detected in cytoplasm and very close to nucleus. Moreover, in cases of CP and CEP with inflammatory infiltration, there were a markedly stronger IL-6 expression (+ + + +), than that observed in specimens without infiltrate.CONCLUSION:In conclusion, the results presented herein clearly demonstrated a moderate and strong expression of IL-6 in exocrine and endocrine cells of patients with CP and CEP. This suggests that elevated IL-6 levels of patients with pancreatitis are probably owing to leakage of this cytokine in the circulation following massive pancreatic cells destruction. Recent data suggest that thymus derived CD4+CD25+ regulatory T cells play an important role in the tolerance of the immune system towards tumors. These regulatory cells specifically express the transcription factor FoxP3 which is believed to be a master regulator of regulatory T cell development. In contrast to this thymic development, recent data have demonstrated that CD4+CD25+ regulatory T cells can also be induced from naRve cells in the periphery. We and others have further provided evidence that TGF-beta is critical for this peripheral induction of regulatory cells by inducing the expression of FoxP3. Here we now demonstrate that the induction of FoxP3+ regulatory T cells by TGF-beta is a physiological event in the colon with a potential role in the pathogenesis of colon cancer.Accordingly mice were injected with a single dose of the mutagenic agent azoxymethane followed by three weekly periods of Dextran sulphate (DSS) in drinking water, interrupted by each 14 days of recovery. Treated animals developed numerous tumors in the colon. In a recent study we have shown that tumor growth in this colitis associated colon cancer model was driven by inflammatory CD4+ T cells infiltrating the tumor. As described for human colon cancer, we now demonstrate that dysplastic epithelial cells produced very high amounts of TGF-beta. Interestingly CD4+ cells isolated from the same tumors expressed high levels of FoxP3 mRNA while CD4+ cells isolated from surrounding non-dysplastic colon tissue did not. Our findings were further confirmed by immunohistochemical staining. Accordingly, FoxP3 expressing cells were found in high numbers in the lamina propria of the tumor closely associated to dysplastic epithelial cells. In contrast only few FoxP3 positive cells were detectable in the lamina propria of surrounding normal colon tissue. Based on these data, we speculated that tumor derived TGF-beta may induce FoxP3 in tumor infiltrating T cells. In order to functionally analyze whether tumor derived TGF-beta may play a role in the induction of FoxP3+ regulatory T cells in vivo we induced colon tumors in mice overexpressing a dominant negative TGF-beta receptor specifically in T cells. Tumors collected from these mice showed a similar infiltration with CD4+ T cells as compared to wildtype mice. However, CD4+ T cells isolated from tumor tissue of such transgenic mice showed strongly diminished expression of FoxP3 mRNA as compared to tumors of wildtype animals. Strikingly, tumors in transgenic mice were larger that wildtype tumors implicating that in this model of inflammation dependent colon cancer, tumor induced regulatory T cells control tumor growth.Based on our findings we propose a model in which tumor derived TGF-beta induces FoxP3 expression in infiltrating CD4+ T cells giving them a regulatory phenotype. Such tumor induced regulatory T cells in the case of inflammation dependent cancer can control tumor growth. However, in spontaneous cancer development tumor induced regulatory T cells may mediate tolerance towards the tumor by inhibiting anti-tumor immunity. Accumulating studies has addressed inflammatory bowel disease as an autoimmune disease. However, it is not known whether the intestinal epithelial cell-derived antigens are involved in generation of mucosal immune responses or are the target of the pathogenic process. By using a modified Serological Analysis of Recombinant cDNA Expression Libraries (SEREX) which is an antigen-screening approach utilizing humoral and cellular immune responses, we herein identify an epithelial cell-derived endogenous lectin, galectin-4 (G4), as a pathogenic mediator to exacerbate intestinal inflammation. G4 specifically stimulated the production of IL-6 by CD4 + T cells but not other cell types present in the diseased colon of CD45RB high -treansfer and DSSinduced colitis models and TCRa knockout (KO) mice. In contrast, G4 was unable to stimulate IL-6 production by CD4 + T cells present in the normal colon of these colitis models as well as wild type mice. Confocal microscopic analysis showed that G4 interacts with the immunological synapse on colonic CD4 + T cells as indicated by specific binding of G4 to the lipid raftaccumulating regions. Interestingly, the binding intensity of G4 on CD4 + T cells and the G4-mediated IL-6 production were increased under intestinal inflammatory conditions. Indeed, expressions of a member of sialyltransferases (ST6GalNAc-2, 3, 6 and ST8Sia-1,3) that are specifically utilized for the modification of O-glycans were markedly downregulated in the CD4 + T cells only under inflammatory conditions. In addition, the exposure of core1 Oglycan without sialylation on the CD4 + T cells was confirmed by intensified binding of PNA that specifically binds to this oligosaccharide structure. Mechanistically, the G4-mediated IL-6 production by CD4 + T cells is mediated by protein kinase C (PKC) u-associated cascade as indicated by a fact that colonic CD4 + T cells from PKCu KO mice treated with DSS were unable to respond to G4 to produce IL-6. Functionally, administration of anti-G4 mAb not only led to the attenuation of chronic colitis in B cell-deficient TCRa double knockout mice but also effectively enhanced the recovery from 3.5% DSS-induced acute colitis. These studies not only indicate the presence of an immunogenic epithelial lectin that contributes to the exacerbation of intestinal inflammations but also provide a novel insight into the biological role of lectin/CD4 + T cell interactions under inflammatory conditions. Immunohistochemical Localization of Copper/ Medicine, Wroclaw, Poland; 3 Department and Clinic of Gastointestinal and General Surgery, Wroclaw University of Medicine, Wroclaw, Poland.We found only scare acinar cells staining positively for Cu/Zn SOD in the normal human pancreas (the body and tail of pancreas) (À/+; +); islets cells did not show Cu/Zn SOD immunoreactivity. In slices of the pancreas, derived from patients with CP and CEP, a much stronger immunohistochemical reaction (+ +; + + +) was noticed as compared to controls. Cu/Zn SOD was localized in both acinar and islet cells of the pancreas. Interestingly, immunohistochemical reaction of ducts cells was considerably stronger (+ + + +) than that of islet and acinar cells (+; ++). We also compared expression of Cu/Zn SOD and metallothionein (MT), at the same histological specimens and experimental conditions. Whereas, Cu/ Zn SOD was markedly manifested in ducts cells of pancreas, MT did not apeared in it.CONCLUSION:In conclusion, these studies clearly demonstrate a moderate and strong expression of Cu/Zn SOD in acinar, islets and duct cells of patients with CP and CEP. The overexpression of this enzyme in ducts cells may function as an intracellular antioxidant and can compensate for the lack of MT in the cells of pancreas. Peptic fragments of gliadin were shown to activate cells of innate immunity including macrophages/monocytes to cytokine and chemokine production. The aim of this study was to examine whether: (i) peripheral blood monocytes (PBMoC) respond to peptic digest of gliadin by IL-8 and or TNF-a production, (ii) this activity depends on the presence of IFN-g, (iii) there exist a difference in response of PBMoC isolated from blood donors, active and treated (GFD) coeliac patients (including analysis of the causal factors) (iiii) the signalling pathway is mediated via NF-nB molecule activation.Methods: PBMoC were incubated with various doses of peptic digest of gliadin alone or together with IFN-g, or pre-incubated with IFN-g before adding the gliadin. HLA genes were typed using PCR with sequence-specific primers (SSP-PCR). NF-nB DNA binding activity was detected by TransAM NF-kB transcription factor assay kit.Results: The capacity of monocytes isolated from active CoD patients and patients on GFD to produce IL-8 was significantly higher than that of healthy donors. The simultaneous addition of IFN-g had no enhancing effect on IL-8 production and the prestimulation of cells with IFN-g for 24 hours resulted in a significant increase of IL-8 production mainly in cells from healthy controls. The enhanced TNF-a secretion was detected mainly in gliadin stimulated monocytes from CoD patients and was markedly increased by simultaneous addition of IFN-g. Interestingly, prestimulation of cells with IFN-g for 24 hours increased gliadin-induced TNF-a production in the group of healthy donors and patients on a GFD, and slightly in the group of active CoD patients. This effect reduced the differences in TNF-a production among tested groups. The signaling pathway triggered by gliadin was mediated via NF-nB subunits p50 and p65. Specific inhibitors suppressed DNA binding activity of NF-nB as well as gliadin induced IL-8 and TNF-a secretion. The impact of HLA-DQ2/DR3 antigen expression in healthy donors and the keeping of GFD in treated patients on cell response were evaluated.Conclusions: IL-8 and TNF-a produced by the cells of innate immunity could enhance the effect of gliadin specific lymphocytes and participate in the cascade leading to the damage of intestinal mucosa in celiac patients. Objective: EE is an allergic inflammatory disorder of the esophagus with a significant recent increase in the number of reported pediatric cases. The initiation of the esophageal inflammatory injury has been theorized to be due to food allergies and possibly aeroallergen sensitivities. We reviewed the cases of 32 pediatric patients (pts) with EE in order to discern clinical, histological, and treatment correlates and to acquire a better understanding of disease pathogenesis.Methods: Allergy (food and aeroallergen) testing was completed on 26/32 pts. Serial Esophageal Biopsies were performed on 23/32 pts and serial clinical scores were determined on 25/32 pts. Clinical scores (0 to 5) were based upon the presence/absence of Abd pain, Vomiting, Dysphagia, Wt loss/gain, and Chest pain.Results: 22/25 pts (88%) demonstrated clinical improvement based on a comparison of clinical scores from baseline until after 3 to 45 months of treatment. Treatment: 11/22 pts-Elimination Diet (ED) + Steroids (swallowed Flovent or oral steroid); 8/22 pts-ED only; 2/22 pts-steroids only; 4/22 pts-noncompliant or PPI only.While the majority of pts (65%) demonstrated diminished esophageal eosinophilic infiltration (15/23 pts who underwent serial biopsies), 8/23 demonstrated clinical improvement with no evidence of diminished esophageal eosinophilic infiltration. No differences in treatment regimens existed between these groups. Further, 2/8 pts had been treated longer than average lengths of time, respectively for 30 and 34 months.Conclusions: EE is an allergic inflammatory disorder that in most cases clinically responds to a treatment regimen of ED and steroids. A subset of patients demonstrated an improved clinical outcome without diminished esophageal eosinophilic infiltration. These results may reflect an incomplete therapeutic response which requires close follow-up in order to detect an end of disease remission. Alternatively, these findings may be characteristic of a unique patient subset that merits further investigation. Infection by human immunodeficiency virus can involve several clinical manifestations affecting almost every organ or system in the body. 46 patients (43 males, 3 females), with infection by HIV, from South Italy, were recruited for the study. HIV positivity was detected by enzyme-linked immunosorbent assay (ELISA) and confirmed by Western Blot. Patients were asked to complete a questionnaire containing 10 questions pertaining to rheumatic diseases. The following data were collected: age, sex, duration of HIV infection, CD4 count. Rheumatologic manifestations were found on clinical evaluation by questionnaire administration and physical examination in 11 of 43 patients (23.9%). RESULTS: Arthralgias were the commonest manifestations, occurring in 9 patients (82%). Pain was usually intermittent and of moderate intensity and involved 2 or more joints. One patient had ReiterTs syndrome (9%) with a mild oligoarthritis of the knees and spondylitis responding promptly to NSAIDs. Another patient had a mild oligoarthritis (9%) involving knees and ankles. Quality Control of siRNA, Optimizing ItTs Transfection Efficiency and Monitoring CD4 Gene Silencing Effect with a Microfluidic Chip Device.T. Preckel, 1 C. Buhlmann, 1 M. Valer. 1 1 Liquid Phase Analysis, Agilent Technologies, Waldbronn, BW, Germany.Gene silencing with RNA interference (RNAi) is a new breakthrough technology with high potential for development of therapeutics. Here, the delivery of small interfering RNA (siRNA) into cells is of key importance in elucidating gene and protein function. Differing types of interfering RNAs and several methods of delivery into cell types exist. Furthermore, the selection of the best silencing sequence at optimized siRNA transfection conditions is based on the integrity and purity of siRNA, siRNA uptake and cell viability. Given the complexity of monitoring and optimizing these types of experiments a new tool is required that would allow for minimal sample and reagent consumption in a fast and easy to use format.We describe the use of a microfuidic chip-based system to quickly verify siRNA quality and to determine the optimal conditions for gene silencing experiments. First, RNA integrity and purity is assessed. Second, fluorescently labeled siRNA is used to optimize transfection parameters in mammalian cells. Life cell staining is performed on-chip reducing the overall analysis time for 6 samples to less than 50 minutes from harvesting the cells to final results with actual hands-on-time of less than 10 minutes. Transfection efficiency is measured as the percentage of cells with a strong siRNA uptake within the live cell population. Third, gene knockdown is measured with the same system. Here, staining the protein of interest, e.g. CD4, with fluorescently labeled antibodies demonstrates the downregulation of proteins after siRNA transfection. The gene silencing mechanism can also be verified in a given cell line by using a GFP-tag and cotransfecting the tagged protein and a Cy5 labeled siRNA against that protein. Successful silencing can be measured by reduced GFP expression within Cy5 positive cell population at different timepoints after transfection. 1 1 Pediatric Immunology, Uludag University School of Medicine, Bursa, Turkey.Omenn syndrome is characterized by generalized erythematous skin rash, lymph node enlargement, hepatosplenomegaly, increased serum Ig E levels, eosinophilia, and evidence of severe combined immune deficiency. Patients develop fungal, bacterial, and viral infections. Echocardiographic investigation revealed a rounded structure filling the apex and corpus of the the right ventricle. We investigated for hypercoagulation state and discussed ventricular thrombosis which is uncommon in Omenn syndrome. Common Variable Immunodeficiency (CVID) is a heterogenous group of B-cell deficiency syndromes characterized by hypogammaglobulinemia, impaired antibody production and recurrent bacterial infections. Vitamin A (Vit A), a naturally occuring antioxidant micronutrient, has immunomodulating effect in patients with immunodeficiency, including an influence on cytokine production and lymphocyte growth and functions. Vit A deficiency is associated with a shift from type 2 cytokines to predominantly type 1 cytokines. The aim of this study was to investigate vitamin A levels in CVID patients and the effects of Vit A deficiency on cytokine production.Nineteen CVID patients and 13 healthy controls who attended to the Department of Pediatric Immunology in Uludag University School of Medicine, Turkey, were involved in this study. Serum Vit A, serum neopterin (indicator of chronic inflammatory state), serum type 1 (TNF-alpha, IL-2) and type 2 cytokine levels (IL-4, IL-10) were determined. 9-cis retinal which is Vit A derivative was added to lymphocyte cultures of CVID patients and controls and the effects on cytokine production were investigated.While serum Vit A levels of CVID patients were obviously low in CVID patients (in 75%), serum neopterin levels were higher than control group (in 31%). Elevated serum neopterin levels and low Vit A levels in CVID patients suggests that serum Vit A levels were lower secondary to recurrent infections seen in these patients. Serum IL-4 level was found lower in CVID patients than controls. The results of lymphocyte culture in CVID patients showed that IL-4 was higher in 9-cis retinal stimulated group than unstimulated group. However the expected change on other cytokines was not seen.As a result, our study shows that CVID patients have low serum Vit A levels and this finding correlates with their chronic inflammatory condition and supplementation with Vit A may have role in downregulation of inflammatory responses.Su2.05. Pulmonary Abscess Due to Aspergillus spp. 1 1 Pediatric Immunology, Uludag University School of Medicine, Bursa, Turkey.Chronic granulomatous disease (CGD) is the most common phagocytic disorder. Invasive fungal infections are an important cause of morbidity and mortality in CGD patients, with Aspergillus spp. being the most frequent fungal pathogens.A fifteen-month-old boy with a cavitation in the right upper lobe presented with persistent weight loss, fever, cough and roentgenographic evidence of right upper lobe abscess resistant to antibiotic therapy. A lung biopsy led to the diagnosis of pulmonary aspergillosis. A respiratory burst assay revealed the diagnosis of CGD.He was treated with high doses of liposomal amphotericin B (7/ mg/kg) for an invasive pulmonary Aspergillus nidulans infection. The infection regressed during 12 weeks of treatment to the addition of interferon-gamma. We report a case of HodgkinTs lymphoma developing in a 4.5year-old child with hyper-IgE syndrome. A white girl with asthma, recurrent pneumonia, bronchiectasis, atopic dermatitis, recurrent skin infections and growth retardation. Immunologic evaluation revealed high level of immunoglobulin E (7000 IU/dl) and peripheral eosinophilia.She was found to have normal values for serum IgG, IgM, IgA, sweat chloride test, WBC chemotaxis and serum complement function. The occurance in this patient of HodgkinTs lymphoma suggests that individuals with hyper-IgE syndrome may also be at increased risk for developing premature malignancies, although the precise immunologic defect in this syndrome is still unknown. The purpose of our study is developing of distribution of variants of allele genes HLA DRB1 and IL-4 (on SNP in a promotor site-C-590T) in HIV-infected patients for an establishment of immunological and genetic markers of determination and/ or resistance to HIV-infection.HLA-detecting of HLA DRB1 genes and IL-4 in 30 HIVinfected patients was made using the method of multiprimer amplifications sequence-specific primers basis on PCR at a level of allele groups with the set of reagents: (bDNA-technologyQ, Moscow). Variants of allele gene IL-4 were defined with the help of PCR with the subsequent restriction of products of amplification.Immunological and genetic analysis revealed, that the most significant protective effect have the specificities DR B1*01 (RR = 0,46; PF = 0,15), DR B1*04 (Rr = 0,52; PF = 0,175) and DR B1*13 (Rr = 0,6; PF = 0,108). Quantity of HIV-infected people with homozygous variant C/C was lower, than in healthy European people (28,6 % and 83,3 %, accordingly), while with variants T/T (21,4% and 0%) and C/T (50,0% and 16,7 %) are essentially higher. Besides distinctions in formation of the immune response and cytokine status in HIV-infected patients have been revealed various variants of promotor allele gene IL-4.The received results testify that immunological and genetic factors have essential influence on processes of infecting and development of disease and can be used for defying of probability of case of infection, and the individual predicting of disease, and for the development of the individualized approach for monitoring and diving adequate immunocorrection therapy of the HIV-infection. Hyper-IgE syndrome is a rare primary immunodeficiency of unknown etiology characterized chronic eczema, elevated total serum IgE, recurrent infections of skin and respiratory tract, and variable associated skeletal symptoms. Recent reports about pyogenic bacterial infections in patients and animals with defects in Toll-like receptor (TLR) pathways lead us to search for related defects in patients with hyper-IgE syndrome. Su2 Cytokine profiles in six patients with hyper-IgE syndrome were analyzed in serum samples, in T cells after stimulation with PMA/ Ionomycin and in peripheral blood mononuclear cells stimulated by TLR ligands and bacterial products including LPS (TLR4), peptidoglycan (TLR2), PolyIC (TLR3), R848 (TLR7/8), CpG-A and CpG-B (TLR9), zymosan and heat killed listeriae monocytogenes. Results were compared to healthy controls.Reduced percentage of IFN-gamma, IL-2, and TNF-alpha producing T cells after PMA stimulation in patients with hyper-IgE syndrome was found implying an impaired inflammatory T cell response. Augmented serum levels of IL-5 point to an associated Th-2 shift. However, normal production of cytokines (TNF-alpha, IL-6, IL-10, IFN-gamma, IP-10) and upregulation of CD86 on B cells and monocytes after TLR stimulation ruled out a defect in TLR signaling pathways.In conclusion, a dysbalance in T cell responses of patients with hyper-IgE syndrome was detected as described before, but no indication for an underlying defect in Toll-like receptor pathways was observed. Objective: Common variable immunodeficiency (CVID) is a heterogeneous immunodeficiency with characteristic recurrent bacterial infections due to hypogammaglobulinemia and incapacity to generate memory B cells. In addition to dysfunctional immunoglobulin production, patientTs T-cell activation is impaired, resulting in low production of cytokines and decreased T-cell proliferation. Patients further suffer from gastrointestinal infections, autoimmune disease, and various B-cell neoplasm. We addressed whether CVID is associated with impairment in the dendritic cell (DC) compartment, as DCs play a central role in the development of adaptive immunity. Materials and Methods: Heparinized blood samples were collected from CVID patients at least 21 days following the last infusion of intravenous immunoglobulin (IVIg) or from newly diagnosed naive CVID patients prior to IVIg therapy. As control, blood samples were obtained from patients with selective antibody deficiencies who received IVIg similar to CVID patients, and healthy controls. Monocyte-derived DC from patientsT blood and from control groups were generated after differentiation for six days in the presence of GM-CSF, IL-4 and10% autologous plasma.For allogeneic mixed lymphocyte reaction, DCs from CVID patients and healthy donors were exposed to CD4 + T-cells of thirdparty healthy donors.The CD4 + T cells were isolated by magnetic cell sorter (MACS) cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany). Cytokines were quantified by Quantikine ELISA kit (Immunotech, Marseilles, France).Results: Dendritic cells (DC) from CVID patients have severely perturbed diiferentiation and maturation. Although the total number of DC appears normal as determined by CD11c expression, they express only nominal levels of CD1a, a hallmark of LangerhanTs cells, and completely fail to up-regulate CD83, typically expressed on fully mature DC. Moreover, patientsT DC express markedly reduced levels of the costimulatory molecules CD80 and CD86 that are critical for T-cell stimulation; and HLA class II, the antigen presenting molecule. In turn, patientsT DC induced weak proliferation of allogeneic T cells and produced significantly low amounts of interleukin-12 (IL-12) upon CD40 signaling.In our study 75 children with otitis media and 75 healthy children under age of 6 year were evaluated. Serum gamma globulins were determined with serum electrophoresis. IgM, IgG, IgA, C3,C4 in serum were determined with single radial immunodiffusion.Level of IgG 2 in serum was determined with Sandwich ELISA through our Homemade ELISA.Result: Show that otitis media occurred in male 1.3 times more than female. The level of gammaglobulins,IgM,IgA have increase in patient group versus health group in 1-2 year age group (P b 0.05), but no meaningful increase in 3-4 and 5-6 years age group was noted (P N 0/05). No statically difference was noted in the level of IgG,C 3 and C 4 between patient and health groups in all age groups (P b 0.05). IgG 2 has decreased in group versus health group in 3-4 year age group (P b 0.05) but no statistical difference was noted in 1-2 year and 5-6 year age groups (P N 0.05).Su2.28. New Composition Immunotherapy in Treatment of Secondary Immunodeficiency Accompanied by Viral Infectious Syndrome. Among different clinical syndromes accompanied the secondary damage of immune system-the viral syndrome is leading one. Our studies last years had demonstrated facts: persons having few recurrent episodes, from 4 till 24, of acute viral respiratory infections (RAVRI) by the year may have also persisting viral infections such as CMV, VEB, HSV (I, II types), VHG-6. All of them had clinical markers of secondary immunodeficiency accompanied by viral infection syndrome. We detected: the system of IFN (plasmaTs IFN, IFNa, IFNg); immune system (CD3+, CD4+, CD8+, CD16+, CD19+, RBTL, IgG, IgA, IgM, neutrophylic phagocyts); herpes viral infection (HSV I/II types, CMV, VEB, VHG-6) by means of PCR and there were tested specific antibodies-IgG and IgM classes against HSV I/II types, CMV, VEB. 100% patients had have combine defects of IFNTs system, different deficiencies of T-cellTs immunity, NK cells, phagocytes. More than 90,0% patients had have suggesting herpesviral infections. All of this patients were treated following complex immunotherapy in different combination: 1) local and systemic IFN therapy (recombinant IFNa-viferon-1, -2, -3, -4, viferon oil), using high-, middle-and low-dose therapy during 2,5-3,5 month-base therapy; 2) specific and non specific passive intravenous immunoglobulins (cytotect, pentaglobin, intraglobin, octagam) only if it was necessary; 3) immunomodulation therapy thymusTs factors (tactivin, tymogen, imunofan) was directed to restoration of T-cellTs immunity; 4) synthetic preparation with polyvalent effects as polyoxidonium and licopidum (reconstruction of NK and neutrophylic leucocyts). At present itTs possible to combine immunotherapy and therapy with synthetic antiviral drugs. This kind of immunoreconstruction may be accompanied by bsupport therapyQ. Using new kinds of complex immunotherapy in treatment of secondary immunodeficiency accompanied viral syndrome we had obtain positive clinical and immunological effects. Those effects had shown decreasing frequency of acute episodes of recurrent viral infections from 4-24 till 1-2 per year, elimination of viral pathogens (81,5% patients), reconstruction of destroy different immune mechanisms (88,8%). Phenotypic Analysis of Peripheral Blood and Tissue Memory B Cells in Common Variable Immunodeficiency (CVID). A. P. Williams, 1 C. Stephens, 1 L. Hedges, 1 A. Mani, 1 E. Hodges, 1 J. Smith. 1 1 Department of Clinical Immunology, Southampton General Hospital, Southampton, United Kingdom.CVID is a heterogeneous primary immunodeficiency disorder characterised by hypogammaglobulinaemia and repeated bacterial infections. Whilst many cellular abnormalities have been described in such patient, recent interest has focused on subtyping such patients based upon the presence or absence of peripheral B cell memory subsets. We have developed a whole blood assay for the detection of naRve and memory B cells in peripheral blood and compared this to the previously described PBMC separation techniques for subtyping peripheral B cells. Additionally, we have analysed a cohort of CVID patients using this assay and compared their peripheral blood memory B cell subset distributions to that seen within their peripheral secondary lymphoid organs.Four colour flow cytometry was undertaken upon lysed whole blood and density centrifugation separated peripheral blood mononuclear cells. A series of 10 healthy controls was used to compare the 2 methods and a further 30 controls analysed to set up a reference range using the whole blood assay. 11 CVID patients were subtyped by this method into MBO (no memory cells), MB1 (no class switched memory cells)and MB2 (normal memory cells) classifications. Patients within the MB0, MB1 and MB2 classifications that had secondary lymphoid tissue archived, were analysed by immunohistochemistry for the presence of memory B cells.The whole blood assay performed well and correlated with the density centrifugation separation technique previously described. For the IgM memory cells the correlation coefficient (R 2 ) between the two assays was 0.958 and for class switched cells it was 0.984. A normal reference range was established using healthy controls. As a percentage of peripheral B cells, naRve B cells = 64% F 24% (2SD), total memory cells = 32% F 24%, IgM memory cells = 17% F 16% and class switched memory cells = 15% F 12%. 11 CVID patients were analysed and 5 subtyped as MBO, 4 as MB1 and 2 as MB2. These subtypes were stable over a 12 month period. Tissue sections were analysed for a representative patient of each of the 3 groups. A lymph node from a MB0 patient showed no evidence of germinal centre CD27 B cells compared to a normal tonsil. Similarly a lymph node biopsy of a MB1 patient showed no evidence memory B cells in a lymph node. Finally, a duodenal biopsy of a MB2 patient did show evidence of CD27 positive memory B cells within this tissue.The whole blood assay appears to be as informative as the previously described assay for assessing B cell memory status. This assay is more cost effective and convenient to undertake within a routine laboratory. The reference ranges establish a minimum level of 12% for total memory B cells (mean F 2SD) with no individual having less than 14% total memory cells, 4% IgM memory cells or 9% class switched memory cells. Such B cell memory subsets are constant over time in adult patients with CVID and correlate with the B cells identified in their peripheral lymphoid tissues. Objective: The recovery of CD4+ T cells in children after HAART supports the existence of some homeostatic mechanism that detects low levels of CD4+ T-cells and activates the necessary mechanisms to achieve a T-cell repopulation. In our experience, the thymus plays a key role in this repopulation, being thus these homeostatic mechanisms likely to lead to an increase in the thymic production of new T cells. In order to establish the role of plasma IL-7 levels before HAART as prognostic marker of CD4+ T-cells recovery in HIV-infected children, a retrospective longitudinal study was performed in HIV-children on first-line of highly active antiretroviral therapy (HAART).Patients and methods: The inclusion criteria were: a) to begin HAART with protease inhibitor; b) CD4+ T-cells V20% at entry to the study; c) at least 6 months on follow-up; d) more than one year of age. From initial 67 HIV-infected children on first-line HAART, only 27 HIV-children with CD4+ T-cells V20% had biological samples and they could be selected to be studied. Those HIVinfected children were divided into two groups according to their percentile 75 (P75) of plasma IL-7: a) Low IL-7: 21 HIV-children on HAART with low IL-7 (IL-7 VP75 (11.97 pg/ml)) at baseline; b) High IL-7: 6 HIV-children on HAART with high IL-7 (IL-7 N P75 (11.97 pg/ml)) at baseline.Results: HIV-infected children with plasma IL-7 levels N11.97 pg/ml achieved CD4+ T-cells z25% faster than HIV-children with plasma IL-7 levels V11.97 pg/ml (P =0.017). This way, HIVchildren with plasma IL-7 levels N11.97 pg/ml achieved CD4+ Tcells z25% to 10.6 (Confidence interval of 95% (CI95%): 0; 23.1) months and HIV-children with plasma IL-7 levels V11.97 pg/ml achieved CD4+ T-cells z25% to 37.1 (CI95%: 4.7; 69.4) months. The RP of immunological response to HAART were also calculated, and high IL-7 group had a RP to achieve CD4+ Tcells z25% of 3.24 (CI95%: 1.16; 9.0).Conclusion: Our data indicate that IL-7 was a good marker of CD4+ T-cells recovery in HIV-infected children. Mathematical modeling the transmission and dynamics of HIV infection can lead to a deeper understanding of the disparate experimental data and identify therapies most likely to be effective in controlling the disease. Dynamically modeling the timedependent HIV prevalence in the Nairobi Sex Worker cohort lead to the prediction, subsequently observed, that the observed resistance or immunity to HIV infection exhibited by some members of the cohort is not absolute but transient. Thus, whatever cellular or other type of immunity that is responsible for this observed resistance to HIV infection is not strong enough to reliably prevent infection. The dynamics of HIV infection below the viral density threshold can only be inferred from a dynamical model extrapolated into this region. Analyzing published modeling results on the steady-state HIV infection in chronically infected patients leads to the conclusion that the viral load doubling times immediately following HAART interruption are virtually identical to the above results for primary infection patients. Moreover, the doubling times at threshold for infected patients treated with vaccine and/or IL-2 therapies before and/or after HAART withdrawal are virtually identical to those of untreated cohorts. Thus, neither prolonged familiarity with HIV infection nor vaccine and/or IL-2 therapies significantly increase the strengths of the immune systemTs humoral or interferon responses to HIV infection. An important question to answer is why vaccine and/or IL-2 therapies all fail to measurably strengthen the immune system response to HIV infection. Dynamically modeling the rebound of HIV infection following HAART withdrawal leads to the conclusion that the immune systemTs inability to reduce the viral load to a value below threshold is not due a failure in its phagocytic arm but to the failure of its cellular arm to clear productively infected cells from the host at a rapidly enough rate. This failure is traceable to the existence of a pool of productively infected CD4+ cells (T cells, natural killer cells, etc.) whose half-lives in vivo are uncharacteristically long so that their numbers are easily replaced by newly infected, similarly long-lived, CD4+ cells. One compartment of this pool consists of CD4+ Natural Killer cells that are HIV-1infectable, have higher survival half lives than typically infected CD4+ T cells, and are unaffected by HAART. This pool cannot be cleared by the immune system, primed by vaccines and/or IL-2 or not, and is responsible for the observed, characteristic, saturation in the viral density curve at the viral set-point. Thus, an HIV infected immune system cannot eliminate all sources of viral production on its own-outside antiviral intervention is needed. Expanding HAART to include fusion blockers and overcoming its current deficiencies is the most promising path to take in seeking an effective therapy for HIV infection. Moreover, achieving and using the ideal HAART as a prophylactic would prevent infection in the uninfected. Model for the Repopulating Capacity of Transplanted T Cells. Sebastian Newrzela, 1 Sanaz Taromi, 1 Roland Zahn, 1 Dorothee Von Laer. 1 1 Georg-Speyer-Haus (Chemotherapeutisches Forschungszentrum), Georg-Speyer-Haus (Chemotherapeutisches Forschungszentrum), Frankfurt AM Main, Hessen, Germany.Transfer of gene-modified T cells is a potent option for treatment of immune deficiencies. A major technical challenge is to preserve the repopulating capacity of T cells during ex vivo expansion and culture.A limited survival of transplanted gene-modified T cells was observed in several clinical trials. This shows that the transplanted ex vivo expanded T cells may have a competitive disadvantage to the non-manipulated cells in the recipient, especially if the transgene does not confer a strong selective advantage.We established a transplantation model in the lymphocyte deficient mouse strain Rag-1À/À to analyze the repopulation capacity of transplanted T cells. Donor T cells were collected from Ly-5.1 C57BL/6 and Ly-5.2 mice. One population was treated with an ex vivo expansion and transduction protocol, the other population served as unmanipulated competitor cells. Both populations were mixed in several variations and transplanted into Rag-1 recipients. The competitive repopulation between treated and untreated cells was analyzed by distinguishing surface antigen expression. The data predict that not only high levels of gene marking, but also the dfitnessT of transplanted cells determine the effectiveness of therapies with gene-modified T cells. BW presented at age 2 yrs with tonsillitis, pneumonia, lymphadenopathy, oral thrush, anaemia and hepatosplenomegaly. Investigations revealed lymphopenia wbc-5.2  10 9 /L (neut: 4.5, lymp: 0.5, mono: 0.1). Throat and mouth swabs revealed a h haemolytic group C strepococcus, pseudomonas aeroginosa and candida, chest radiography showed right middle lobe pneumonia. He was treated with a IV erythromycin and cefuroxime and made a good recovery. Four months later he presented with strepococcal pneumonia treated with intravenous penicillin and cefotaxime and was commenced on long term penicillin prophylaxis. At the age of 3 years he presented with a vasculitic rash on lower limbs and buttocks compatible with pityriasis lichenoides. At this stage he was assessed immunologically, this revealed normal serum IgG (13 g/l), IgM (6 g/l) and IgA (1.7G/L) and a poor response to pneumococcus and HIB vaccinations. Immunophenotyping of blood lymphocytes showed reduced CD3, CD4 and CD8 positive cells (0.16, 0.07 and 0.09  10 9 /l respectively), normal CD19+ B cells (0.27  10 9 /l) with a poor response to PHA and anti-CD3. EBV serology was positive for IgG consistent with recently acquired infection. An apoptotic defect of both CD4 and CD8 cells was shown with excessive surface expression of CD95 on CD4 and CD8 cells, together with enhanced apoptosis in cell cultures as judged by propidium iodide and annexin V staining. At age 5 yrs BW mounted a good antibody response to pneumococcus and HIB and lymphocyte response to PHA was normal. However, his CD3, CD4 and CD8 lymphopenia (0.24, 0.11 and 0.11  10 9 /l respectively) persisted. BW was monitored routinely and at age 10, his lymphocyte count improved and a population of CD4 CD8 dual positive cells was found for the first time (lymphocytes 1.2 Â10*9/l, CD3 0.76, CD4 0.24, CD8 0.18, CD4 CD8 dual positives 0.4). A TCRVB7 clone was identified in association with CD4 CD8 double positive cells confirmed by cell sorting analysis. This clone was not identified in earlier populations of cells and there was no evidence for TCR or IgH clones in earlier cell preparations. BW is currently well without prophylactic antibiotics. We present a child with an apoptotic defect and low CD4 count who developed a clonal CD4CD8 dual positive TCRVB7 T cell population with clinical improvement. While there is no evident cause for this population, viral aetiologies remain a possibility. spanning 1.4 cM were used to determine the haplotypes. Eight haplotypes were observed more than once across the families (47 % of all cases). Twenty eight different mutations were identified in 46 patients, 14 of which were new; most were aberrant splicing. In all, eight founder mutations were found in the 92 alleles. Phenotypic features of the patients (presence of ATM protein, ATM kinase activity, CSA, serum AFP level, serum Ig levels, antibody production against pneumococcal polysaccharides, frequency of sinopulmonary infections, rate of progression of ataxia and outcome) were compared to genotypes. In general, disease outcome did not correlate with genotype; however,differences in disease progression were noted. Patients with mutation 3576GNA had milder disease with infrequent sinopulmonary infections, longer life span, and positive antibody production against pneumococcal polysaccharides. Five of the 57 patients developed lymphoma, two developed leukemia and one developed thyroid carcinoma. This survey demonstrated that haplotyping is an efficient initial screening technique to identify mutations in Turkish AT patients and other ethnic populations. Elena Dorofeeva, 1 Roman Khanferyan. 1 1 Immunoregulation, Institute of Allergy and Asthma, Krasnodar, Russian Federation.Background. The fungi have been implicated in inflammatory reactions with immune and non-immune mechanisms, such as rhinitis, asthma, hypersensitivity pneumonitis, allergic bronchopulmonary mycoses, allergic fungal sinusitis, extrintic allergic alveolitis etc. The exact prevalence of fungal allergies is not known. Studies based on skin tests suggest that at least 3-10% of adults and children worldwide are affected by fungal allergy (Bush RK, Portnoy JM, 2001; Homer WE et al., 1995) . Apart from their direct allergenic effects, fungi may carry mycotoxins in their spores or produce volatile metabolites (Miller JD et al., 1998) . Trichotecene mycotoxins are a group of structurally toxins produced mainly by Fusarium fungi found on many crops. A variety of Fusarium fungi produce a number of different mycotoxins of the class of trichothecenes. Three of the better known toxins are T-2, HT-2 toxin and deoxynivalenol (DON, vomitoxin) . Inhalation of mycotoxins such as aflatoxins, secalonic acid D, zearalenone and trichothecens produced by Aspergillus, Penicillium and Fusarium fungi, may affect the immunological response of the lung tissue or present other hazards to human health.Aim of investigation. To evaluate the possible mechanisms of immunosuppressive effects of T-2 toxin and DON on different cells and mechanisms of immune system.Results. Both mycotoxins dose-dependently inhibited stem cell proliferation in mice and PHA-induced MNC proliferation. T-2 toxin and DON suppress the activity of peritoneal, spleen, bone marrow and alveolar macrophages. The production of IL1 under the influence of toxins decreased as well as the activity of different hydrolases. Most sensitive were enzymes in bone marrow macrophages. T-2 toxin as well as DON suppressed the activity of all enzymes, especially thiol proteinases-catepsins B, C and H. Less sensitive were hydrolases of spleen and peritoneal macrophages.Conclusion. T-2 and DON trichotecenic mycotoxins induce immunotoxic, mainly immunosuppressive effects on different immune cells via multiple mechanisms. Bone marrow cells are most sensitive to immunosuppressive effects of studied mycotoxins. Immunotherapy by Cytogenes in Patients with Acquired Neutropenia. Vardan Sergey Aprikyan, Nata Adolf Otieva. of Biochemistry, Yerevan, Armenia; 2 Pharmacology, State University, Yerevan, Armenia.It is known that acquired neutropenia is disorder of neutrophil production predisposing patients to acute or recurrent bacterial infections.The purpose of our study was to examine the effect of new cytogenes-synthetic short peptides on the base of naturally originated brain and bone marrow immunoregulatory peptides, designed in our laboratory on blood count and the development of infections in patients with acquired neutropenia of different genesis. 37 healthy volunteers with previous cytotoxic chemotherapy or radiotherapy were under daily observation. Healthy individuals without cytogenes treatment or with normal blood counts served as controls.It was found that peptides completely restored quantitative and qualitative index of blood neutrophil counts, and stimulated their functional activity. During all time it was not observed any complications deviation of blood count. Only 2 of 37 patients, observable during 4 months after immunotherapy had been illness with the easy form of a simple herpes virus.The results of the presented study indicate that cytogenes may to restore differential blood count, avert the development of infections and to put in complete recovery in patients with acquired neutropenia of different genesis. Introduction: Acute otitis media is acute inflammation of middle ear with signs and symptoms such as fever, otalgia,redness of tympanic membrane and accumulation of fluid in the middle ear. The most important bacteria that cause otitis media are streptococcus pneumonia and hemophilus influenza, both of them have polysaccharide capsule. this along with frequent complications of otitis media encourage us to evaluate the level of immunoglobulin and complement in patients and help them if there is defect in immunity of them. Therapeutic Efficacy of Gc Protein-Derived Macrophage Activating Factor for HIV-Infected/AIDS Patients.N. 1 1 Molecular Immunology, Socrates Institute for Therapeutic Immunology, Philadelphia, PA, USA.Although human immunodeficiency virus (HIV) is reported to be infective to macrophages, the activated macrophages do not support growth of HIV due to lack of DNA replication. Macrophages, when highly activated via inflammation, can recognize (development of receptors for viruses) and destroy a variety of viruses and their infected cells. Inflammation-primed macrophage activation process is the principal macrophage activation cascade that requires serum vitamin D 3 -binding protein (known as Gc protein) and participation of B and T lymphocytes. Stepwise hydrolysis of Gc protein with the membranous h-galactosidase (Bgl i ) of inflammation-primed B cells and the Neu-1 sialidase of T cells yields a potent macrophage activating factor, the protein with N-acetylgalactosamine as the remaining sugar (Yamamoto 1996 . Thus, Gc protein is the precursor for the principal macrophage activating factor (MAF). However, the MAF precursor activity of serum Gc protein of HIV-infected patients was lost or reduced because Gc protein is deglycosylated by serum a-N-acetylgalactosaminidase (Nagalase) secreted from HIV-infected cells (Yamamoto et al. Thus, deglycosylated Gc protein cannot be converted to MAF. Since macrophage-activation for enhanced phagocytosis and antigen presentation to B and T lymphocytes is the first indispensable step in both humoral and cellular immunity development, lack of macrophage activation leads to immunosuppression. Exogenously given MAF, however, can bypass the deglycosylated Gc protein and directly act on macrophages for activation. Stepwise treatment of highly purified Gc protein with immobilized h-galactosidase and sialidase generates the most potent macrophage activating factor (termed GcMAF) ever discovered but it produces no side effect in humans. Efficacy of GcMAF for several HIV-infected/AIDS patients was assessed by HIV-specific serum Nagalase activity because the serum Nagalase level is proportional to the amount of HIV-infected cells (or virus load). After approximately 16 weekly administrations of 100 ng GcMAF (once a week intramuscular injection), these patients exhibited insignificantly low serum Nagalase levels equivalent to healthy controls. Eradication of HIV and HIV-infected cells was confirmed by complete clearance of viral antigens (i.e., 24p and gp120) in their blood stream. The disease did not relapse four years after completion of GcMAF therapy. 1 Molecular Immunology, Socrates Institute for Therapeutic Immunology, Philadelphia, PA, USA.Serum vitamin D 3 -binding protein (known as Gc protein) is the precursor for the principal macrophage activating factor (MAF). The precursor activity of serum Gc protein was lost or reduced in HIV-infected/AIDS patients. These patient sera contained a-Nacetylgalactosaminidase (Nagalase) that deglycosylates serum Gc protein (Yamamoto et al. Deglycosylated Gc protein cannot be converted to MAF, thus loses the MAF precursor activity. Since macrophage activation for enhanced phagocytosis and antigen presentation to B and T lymphocytes is the first indispensable step in both humoral and cellular immunity development, lack of macrophage activation leads to immunosuppression and opportunistic neoplasm. Therefore, the Nagalase activity level of HIV-infected/AIDS patient sera regulates the degree of immunosuppression. In fact, Nagalase was inducible by treatment of HIV-infected patient peripheral mononuclear cells with a provirus inducing agent, Mitomycin C. This glycosidase should reside on an outer envelope protein capable of interacting with cellular O-glycans. Although cloned gp160 exhibited no Nagalase activity, treatment of gp160 with 0.01% trypsin for 30 min expressed Nagalase activity suggesting that proteolytic cleavage of gp160 to generate gp120 and gp41 is required for Nagalase activity. When cloned gp120 was treated with 0.001% trypsin for 30 min, Nagalase activity further increased significantly. This suggests that a proteolytic cleavage site of V3 loop of gp120 may be required for further enhancement of Nagalase activity for infectivity. Therefore, the Nagalase appears to play dual roles in viral infectivity and immunosuppression. Clinical and Laboratory Findings in Twenty Eight Patients with a Primary Immunoglobulin Deficiency Associated with Lymphadenopathy, Hepatosplenomegaly and Pulmonary Infiltrates. Tissue biopsy shows the presence of sarcoid-like granulomas.Objective: To evaluate the underlying pathology of the lymph nodes, liver or lungs in patients with humoral immunodeficiency. The pathology will be correlated with clinical presentation, immunoglobulin levels, T and B-cell counts, and microbiology studies.Design: Retrospective chart review of patients evaluated at the Primary Immune Deficiency Clinic, Mayo clinic Rochester between 1998 and 2004.Setting: Large tertiary care medical center. Twenty eight patients were identified out of a total of one hundred and fifty with a humoral antibody deficiency. The electronic charts of the 28 patients were reviewed.Evaluation: Patients underwent clinical and peripheral blood laboratory studies. The results presented are based on pathologic evaluation of lymph node, lung or liver biopsies. Tissues from biopsies and bronchioalveolar lavage were also cultured for the presence of bacterial, fungal and mycobacterial infections.Results: Sixteen of the 28 patients were males. The average age of the 28 patients was 43 F 14, with a median of 44 and range from 16-23. Generalized adenopathy affected 89%, splenomegaly 71% and hepatomegaly 50%. 50% had sarcoid-like granulomas and 40% follicular lymphoid hyperplasia. Two patients developed lymphoma and one chronic lymphocytic leukemia. One patient had nocardia lung infection, another atypical mycobacterium, one osteomyelitis and two sepsis. Twelve (43%) had a significant autoimmune disease; this includes hemolytic anemia (21%), idiopathic thrombocytopenic purpura (32%), and inflammatory arthritis (7%). Other autoimmune disorders included Celiac disease, systemic lupus, type-1 diabetes, hypothyroidism and anti-phospholipid syndrome. All patients with autoimmune disease had adenopathy, 93% splenomegaly, 57% granulomas and 50% lymphoid hyperplasia. Compared with 75% of those without autoimmunity having adenopathy, 47% splenomegaly, 40% hepatomegaly, 33% lymphoid hyperplasia and 27% granulomas. Patients with granulomatous disease had significantly higher hepatomegaly (P = 0.044) and hemolytic anemia (P = 0.035). Average CD-19 cell count was 217 with a median of 83. No subgroup difference in immunoglobulin levels or T and B-cell counts was noted.Conclusion: Adenopathy and/or hepatosplenomegaly in patients with immunoglobulin deficiency are not all granulomatous in nature but also include nodular lymphoid hyperplasia. Autoimmune disease is also prevalent among those patients. No difference in the immunoglobulin levels or the T and B-cell count between the two groups is noted. Larger number of patients and genetic mutation analysis might differentiate between the different subgroups of patients. The IFNg/IL-12 pathway plays a critical role in controlling mycobacterial infections. Patients with mutations in this pathway show an exquisite susceptibility to these germs. Thereafter, and until he was 2 years old, he presented with recurrent lung infections partially responsive to antibiotics. FP received multiple antimycobacterial drug regimes without clearance of mycobacteria or clinical improvement. At 9 years of age he was referred to our unit. He presented with a mild pulmonary insufficiency, and mycobacterial suppurative cervical adenitis. Traditional primary immunodeficiencies and HIV were ruled out. His sputum persisted culturepositive for another 6 years despite combined anti-mycobacterial treatment (enteral route; according to mycobacterial susceptibility), plus rIFNg (upon availability). IL-12Rh1 deficiency was con-firmed at age 14 (homozygous deletion-insertion GC-NTT 1687-1688; leading to V541V-Q542X). At the age of 15, FP showed a profound clinical deterioration with severe respiratory insufficiency and a 10Â5Â5 cm thoracic mycobacterial mass. The patientTs sputum and mass grew a multidrug resistant M. bovis-BCG (resistant to isoniazid, rifampicin, pyrazinamide, rifabutin, and ethionamide), and his serum showed no mycobactericidal activity in vitro. Protein-loosing enteropathy and steatorrhea was also detected at that time. Two months after this therapeutic scheme was started, his sputum turned culture-negative and his serum showed a positive bactericidal activity in vitro. At 8 months of treatment he presented with a sensitive peripheral neuropathy and optic neuropathy. Because of this complication, streptomycin and linezolid were withdrawn, and capreomycin was introduced. After 1 year of anti-mycobacterial treatment, the whole regime was suspended. Four months later, and under weekly azythromycin prophylaxis, the patient remains culture-negative (14 months), the thoracic mass has completely resolved, the peripheral and optic neuropathy show a significant but not complete recovery, and he leads a normal life. A combination of parenteral and enteral anti-mycobacterial treatment, including linezolid and rIFNg, helped to clear a 15 years-long/13 years culture-positive disease caused by M. bovis-BCG with acquired multidrug-resistance, in an IL-12Rh1 deficient patient with impaired intestinal absorption. For immune monitoring studies during HIV vaccine clinical trials, whole blood specimens from HIV seropositive patients may be collected at multiple sites and sent to a central location for PBMC isolation, cryopreservation and evaluation. Clinical researchers prefer to use cryopreserved PBMC because they enable batch analysis of multiple time points, and the samples can be archived for future evaluation. Typically, investigators have two sample collection options: (1) whole blood can be collected in a VACUTAINERR tube and sent to a central location for isolation of PBMC using Ficoll-Hypaque density gradient separation, or (2) whole blood can be collected in a VACUTAINERR CPTk (Cell Preparation Tube), and PBMC isolated by centrifugation at the collection site, and shipped to a central location in the collection tube. The CPT option has some advantages over the Ficoll-Hypaque option such as ease of use, and less hands-on sample manipulation. For functional studies, it is desirable to know that PBMC samples are handled in a manner that will not compromise the ability of the cells to respond to activation stimuli. It is likewise important that sufficient recovery and viability of PBMC be retained in cryopreserved preparations. Therefore, it would be of value to know that PBMC collected and processed by either method perform in a similar manner. In this study we show a comparison of both PBMC collection options, using whole blood specimens from 19 HIV seropositive patients (CD4 N 350, viral load b 50). The performance of samples collected by these two methods was compared by assessment of antigen-specific CD8+ and CD8-T cell immune responses (using cytokine flow cytometry), and cellular viability and recovery (using a hemacytometer and Trypan Blue exclusion). Antigen-specific IFNg expression was measured by stimulating PBMC with CMV pp65 and HIV p55 peptide mixes for 6 hours in vitro, followed by fixation, permeabilization, and intracellular staining. For data comparison, non-parametric paired t-test analysis of the intracellular expression of IFNg, viability, and cellular recovery was performed. The results indicate that after PBMC had been frozen and thawed, samples tested for CMV-and HIV-specific IFNg expression performed comparably to fresh PBMC under both collection conditions. The viability was significantly different between fresh and frozen PBMC derived using Ficoll-Hypaque (p = 0.0005), although it was never less than 90% for PBMC harvested by either collection method. There were no significant differences in the post-freezing IFNg response, viability, or recovery between PBMC derived by Ficoll-Hypaque and by CPT, suggesting that either collection method would result in comparable yields of the viable cells needed to perform equivalently in functional studies. Overall, these data suggest that CPT is an efficient system for the collection and cryopreservation of functionally active HIV seropositive PBMC, as well as a viable alternative to Ficoll-Hypaque. Background: HIV-1 infection ultimately results in impaired specific immune function by virtue of the initial binding of the HIV-1 virion envelope glycoprotein, gp120, to the CD4 receptor. EGCG inhibits HIV-1 replication in human peripheral blood mononuclear cells in vitro by inhibiting the biochemical activity of HIV-1 reverse transcriptase resulting in a subsequent decrease in HIV p24 antigen concentration. We present evidence that EGCG (at physiologically relevant levels) binds to the CD4 molecule at the gp120 attachment site, thus establishing the potential use of EGCG as a therapeutic treatment for HIV infection. Methods: CD4 + T cells were positively selected by immunomagnetic separation from platelet-depleted human leukopaks to obtain a highly purified CD4 + Tcell population. Nuclear magnetic resonance (NMR) spectroscopy and saturation transfer difference (STD) experiments examined the binding of EGCG to CD4. Inhibition binding studies were assessed by flow cytometry. In STD experiments, addition of 21 mM CD4/binding site to 520 mM EGCG showed strong saturation at rings B and D of EGCG, while catechin or IgG had little effect. To investigate whether the binding of EGCG to the CD4 molecule on human lymphocytes is capable of inhibiting the binding of gp120 to CD4, we analyzed the binding ability of gp120 to the EGCG-treated and untreated CD4 + T cells. EGCG markedly inhibited the binding of gp120 to CD4 + T cells in a dose-dependent manner (0% at 20nM, 25% at 200nM P b0.01, and 45% at 2000nM, P b 0.01). Thus, at physiologically relevant levels of 200nM, EGCG exerted an inhibitory effect. The control catechin did not alter the binding capacity of gp120. Molecular modeling studies suggested a binding site for EGCG (Phe 43, Arg 59, Trp 62) in the D1 domain of CD4, the pocket that binds gp120. It is reasonable to suspect that EGCG has potential use as adjunctive therapy in HIV infection. The potential competitive binding properties of EGCG for the CD4 binding sites by gp120 may translate to an HIV-1 preventative strategy. We evaluated distinct immunological parameters to predict the natural outcome and highlight their relevance in the pathogenesis of this disorder. B cells subsets, stimulated in vitro immunoglobulin production (sIVIP) and specific antibody response to tetanus toxoid, HiB and Streptococcus pneumoniae vaccines have been evaluated in 15 patients (mean age 12-36 mo) with a) IgG values b2DS below the mean for age associated to low values of IgA and/or IgM; b) intact cellular immunity; c) absence of clinical and immunological signs of other immunodeficiencies. No abnormalities in distinct B cell subsets have been documented except for 1/15 patients with a low expression of B memory unswitched cells (CD22+/CD27+/IgD-/IgM-) compared to agematched normal controls (1.8% versus normal range of 6.9-11.2%). Stimulated in vitro immunoglobulin production (sIVIP) in baseline conditions and after addition of IL-10 revealed a normal response to all isotypes in 8/15 (53%) patients, while low values of sIVIgGP, sIVIgMP, sIVIgAP were identified respectively in 6/15 (40%), 3/15 (20%) and 3/15 (20%). sIVIP patients didnTt correlate with serum Ig concentrations. Antibody response to vaccine antigens showed a low antibody response to Streptococcus pneumoniae in 2 children with altered sIVIgGP. These preliminary data emphasize the role of sIVIgGP and sIVIgAP as biological predictive and/or prognostic markers of this disorder and make us advance the hypothesis that a defect of class-switching mechanism can contribute to the pathogenetic scenario of THI. Extensive clinical and immunological evaluation is needed to better define this condition.In contrast susceptibility to the multisystem or hepatotoxic reactions to Nevirapine are associated with the MHC class II region. HLA-DRB1*0101 was associated with such hyper-sensitivity but specifically in those with more than 25% CD4+ cells (OR 17.7; Pc = 0.0006), suggesting the HLA class II molecules are likely to be directly involved in the pathogenesis.Thus immunogenetics laboratories are likely to be involved increasingly in pharmocogenomic studies, both in providing a practical clinical application in preventing drug hypersensitivity reactions and for providing insights into the pathogenesis of immunologically mediated drug hypersensitivity reactions. Multisystem Disease in CAEBV Infection. At the age of 5 he developed uncomplicated glandular fever. He remained well until 9 years of age, when he represented with a history of headaches, hemiplegic migraine and splenomegaly. Ultrasound scan confirmed splenomegaly without hepatomegaly or lymphadenopathy. One year later he developed a left sided BellTs palsy and abnormal optic disc appearances. Ophthalmic review diagnosed bilateral pseudooptic disc swelling with retinal periphlebitis. At the age of 12 his height had fallen from the 10 th centile at age 5 to the 0.4 th centile and an endocrinology assessment was undertaken. A bone scan showed a bone age delay of 3 years but no other abnormalities.At the age of 15 he was admitted with a 2 week history of pyrexia, lethargy and hepatosplenomegaly and an immunology opinion was requested. Investigations revealed 7000 EBV DNA copies per ml with serology consistent with past infection. Liver biopsy showed no evidence of lymphoma but some evidence of acute necrosis and chronic inflammation.Immunological testing revealed hypergammaglobulinaemia with an IgG of 19.3 G/L (normal 6-16) and normal IgA and IgM. Anti neutrophil antibodies were detected but no other autoantibodies were identified. Cellular immunophenotyping was as shown below: CD3 = 1.91 (0.8-3.5) , CD4 = 0.75 (0.4-2.1), CD8 = 1.14 (0.2-1.2), CD19 = 0.15 (0.2-0.6) and NK = 0.08-1.2). Currently his immunophenotype shows a CD3 = 1.12, CD4 = 0.6, CD8 = 0.47, CD19 = 0.02 and NK = 0.05.TCR h immunophenotyping showed an expansion of Vh17 T cells, accounting for 40% of the T cells. This population was mainly CD8 positive (N95%). TCR h clonality studies were consistent with a clonal population. These changes persisted after clearance of EBV which occurred by day 20, following treatment of the acute illness with Rituximab (anti CD20 MoAb) and intravenous immunoglobulin. Further testing outside of the acute illness has shown a low EBV count of 140 copies/ml in blood, a reduction in Vh17 T cells to 9% and the demonstration of EBV DNA in a liver biopsy.This individual exhibits some of the diverse, multisystem features that have been previously recognised in chronic active EBV infection. The underlying immunological defect of such patients is poorly understood. A clearer understanding of the underlying defects in such patients will advance our understanding of how chronic latent viral infections are immunologically controlled and improve therapeutic approaches to this difficult to manage condition. RATIONALE: Infants presenting with recurrent infections and low immunoglobulins(Igs) lacking evidence of other immune deficiency disorders are often diagnosed with Transient Hypogammaglobulinemia of Infancy(THI). Although THI is considered a primary immunodeficiency, it is not well defined, and long term follow-up not reported. The purpose of this study is to: 1) Characterize infants with recurrent infections and low Igs. 2) Analyze characteristics to correlate them with time elapsed to Ig normalization. 3) Provide long term follow-up. METHODS: Patients evaluated between 1977-2004 were eligible if they:1)Were term born and b24 months at presentation. 3) Produced antibody to diphtheria and tetanus. 4) Had evidence of intact cell mediated immunity. 5) Lacked features of other immunodeficiency syndromes and 6) Had at least one follow-up set of Igs. Forty-nine patients had the following collected: Igs at presentation and follow-up, age at presentation and Ig normalization, and gender. RESULTS: Of those included, 33/49 were male and 16/49 female. When the data was evaluated by gender, it took longer for the females Igs to normalize when compared to males. Twenty-five percent of the males had normalized by time of 0.3 years, while 25% of the females demonstrated normalization at 4 years. Likewise, 50% of the males had normalized at 1.1 years post-diagnosis, where it took the females 10.4 years for 50% to normalize. This difference was statistically significant (P b 0.001). When the data was evaluated with respect to gender and age at diagnosis, it was found that the younger the females at diagnosis the longer time to normalization, but for males the earlier diagnosis the shorter time to normalization. This gender by age at diagnosis interaction was a strong trend (Pvalue = .083). Overall the course of these patients was benign. None had serious infections, however 2 female non-identical twins met criteria for IgA deficiency. Over half (59%) had a history of wheezing and 26% were atopic. Several have been followed to age greater than 20 years. CONCLUSIONS: 1) Females presenting with diminished immunoglobulins during infancy require longer time to normalization compared to males. 2) Females presenting younger take a longer time to normalize 3) Males presenting with low Igs during infancy display a strong trend toward longer time to normalization the older at presentation. 4) Children with decreased Igs in infancy are a heterogeneous population. In many, especially females, the hypogammaglobulinemia is neither transient nor limited to infancy and thus diagnosis of THI can only be made retrospectively. 5) Children with low Igs and demostrated ability to produce specific antibody have a generally benign course and fewer infections as they grew older, but excess atopy. Wiskott Aldrich Syndrome (WAS) is characterized by immune deficiency, thrombocytopenia, eczema, and increased incidence of autoimmune disease. The protein deficient in this syndrome, WASp, is required for actin-based cytoskeletal rearrangement and T cell activation. Recent data suggest that the Tec kinase, Itk, is required for WASp activation and actin polymerization. Additionally, Itk-deficient mice show defective T helper cell differentiation. Regulation of T helper cell differentiation and cytokine production is essential for mounting appropriate responses against pathogens, while dysregulation of these pathways has been implicated as a possible cause of hypersensitivity and autoimmunity. Given the increased incidence of eczema and autoimmune disease in WAS patients, we asked whether WASp might also be involved in T helper cell differentiation and function.We have found that murine WASÀ/À CD4+ T cell cultures exhibit marked reductions in secretion of both the Th1 cytokine IFN-gamma and the Th2 cytokine IL-4. Nonetheless, intracellular staining revealed that WASP-deficient CD4 cells produce normal to elevated levels of IFN-gamma and IL-4. These data suggest that WASp may be involved in the secretion, but not production of IL-4 and IFN-gamma in CD4+ T cells.Surprisingly, following challenge with the Th2 inducing agent, Schistosoma mansoni eggs, WASÀ/À mice show heightened responses including larger pulmonary granulomas and higher production and secretion of Th2 cytokines. Moreover, 30 days post-infectious challenge with the Th1 inducing parasite Toxoplasma gondii, WASÀ/À mice show fewer numbers of brain cysts and increased production and secretion of IFN-gamma. Thus, in response to parasitic challenge, WASp-deficient mice can mount both a Th1 and Th2 response in vivo that may be exaggerated. Together, these findings suggest that WASp-deficiency has complex effects on T helper cell effector function, which may provide insight into the development of autoimmunity in patients with Wiskott Aldrich Syndrome.Su2.43. Cyclic CD4 Lymphopenia and Absence Interleukin-2: A Novel Immunodeficiency Presentation.Ronit Herzog, Joan Berman, Shirley Fung, Arye Rubinstein. 1 Allergy and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA.Introduction: Immunodeficiency with CD4+ lymphopenia in the absence of HIV infection has been described and represents a heterogeneous condition. Interleukin-2 (IL-2) is a potent T cell growth factor produced primarily by activated CD4+ T cells, and its synthesis is tightly regulated at the mRNA level. A closely related cytokine, Inteleukin-15 (IL-15), shared two of the IL-2 receptor (IL-2R) subunits, but utilizes a unique IL-15 receptor a chain (IL-15Ra) instead of IL-2Ra (CD25). Both cytokines appear to generate identical intracellular signals; however, they may have distinct in vivo properties. There are few reports related to altered regulation of IL-2 in primary immunodeficiency in human, and one case of human immunodeficiency arising from a mutation in the IL-2Ra. We report here a novel immunodeficiency accompanied by cyclic CD4+ lymphopenia, undetectable IL-2 and high IL-15. Case report: The patient is a 20 year old male, without underlying HIV infection, with recurrent mucosal candidiasis and cyclic CD4+ lymphopenia (CD4+ b250 cells/ul) since infancy. His mother died at the age 41 from Pneumocystis pneumonia with no HIV infection. Immune evaluation shortly before her death showed an IgG level of 440 mg/dl and a low CD4+ T cell count. The patient presented with recurrent oral and esophageal candidiasis preceded or accompanied by a low CD4+ T cell count and anergy to candida. His clinical course has been stable between the episodes of lymphopenia. In a recent exacerbation the patient presented with dysphagia and oral thrush that resolved with oral fluconazole, but was followed by severe diarrhea and weight loss. Colonoscopy revealed severe descending colon colitis with deep ulcerations. All bacterial, fungal, and mycobacterial cultures were negative. Total CD3+ T cells count was low at 630 cells/ul (63%), CD4+ T cells count was low at 200 cells/ul (24%). IgG response to pneumococcal polysaccharide antigens was normal. Mitogen induced lymphoproliferative response was decreased to Pokeweed mitogen. Repeat total CD3+ T cells count doubled to 1,410 cells/ul, and the CD4+ T cells count increased to 296 cells/ ul. Lymphocyte phenotyping revealed low CD4+CD45RA+ naive T cells at 3%, and proportionally increased CD45RO+CD4+ memory T cells to 17%. The percentage of CD4+CD25+ T cells was constantly low at 3% and 6%, and CD19+CD25+ were undetectable (0%). IL-2 and mRNA for IL-2 were undetectable while IL-15 levels were markedly increased. Conclusions: We present a familial immunodeficiency in a patient with cyclic CD4+ lymphopenia, recurrent mucosal candidiasis and ulcerative colitis. The finding of IL-2 deficiency with a concomitant increase of IL-15 may suggest a compensatory mechanism by which IL-15 mounts incomplete adaptive immune responses through an IL-2-independent pathway with a persistent susceptibility to fungal infections. Novel Severe T-Cell Immunodeficiency Syndrome Involving Absent Thymus, Coloboma, Bullous Dermatitis, Eosinophilia, and Elevated IgE. Objective: To report a novel severe immunodeficiency of absent thymus, coloboma, bullous dermatitis (BD), eosinophilia, elevated IgE, and monoclonal T cell expansion with a CD4+ defect. Methods: 10wk WM with hx of coloboma, eczema and otitis presented with BD and Pseudomonas superinfection involving trunk and eyes. Due to the unusual skin findings and ocular pathogen, an immune assessment was done as delineated in results section. Based on immunofluorescence studies, immunosuppressants were stopped. He then developed eczema herpeticum and recurrent BD, hypertension, HSM, and lymphadenopathy. Despite IVIG and aggressive antibiotic therapy, he deteriorated clinically with HSV-1 viremia, Parainfluenza infection, Pseudomonas bacteremia, respiratory failure, and died with Enterococcus and CONS bacteremia at age16wk. While skin biopsy initially showed nonspecific dermatitis, classic features of pemphigus vulgaris were seen on 2 later biopsies. Direct and indirect immunofluorescence studies for pemphigus were not conclusive and serum was negative for desmoglian Ab. Eosinophilia persisted (8-18%) . Serum Ig levels prior to IVIG were IgG 1070mg/dL, IgA 16.1, IgM 120, IgE 39,900 IU/ml. HIVAb and DNA PCR were negative. NBT and complement screens were normal. Isohemagglutinins were negative with blood type O. Lymphocyte phenotyping showed CD3+CD4+ 8.4%(#538/mm^3), CD3+CD8+ 61.9%(#3963), CD3-CD19+ 20.4%(#1024), CD56+ 4% (#256), CD45RA+ 0%(#0), CD29+ 5.6%(#359), and a CD4:CD8 ratio of 0.14. Mitogen studies 10d off immunosuppressants showed very poor response to PHA, ConA and PWM. Karyotype was 46 XY, indicating neither maternal engraftment nor aneuploidy. Echocardiogram and abdominal ultrasound at age 11wk were normal. EBV PCR and CMV antigenemia were negative. No deletions were found by FISH at 22q11 and 10p13. Two monoclonal T cell expansions were detected by PCR of TCR gamma locus. HSV IgM was present and HSV IgG was not detected until after IVIG was given. Tracheal aspirate silver stain was negative. Autopsy revealed an absent thymus, HSM, and diffuse massive lymphadenopathy with lack of normal architecture. Interfollicular zones were depleted with scattered dendritic cells and plasma cells, few T lymphocytes, rare primary follicles, and increased stromal elements without increase in histiocytes. Conclusion: We describe a severe immunodeficiency syndrome consisting of CD8+ T cell predominance, CD4+ and nave T cell deficiency, absence of thymic tissue, poor mitogen responses, 2 distinct monoclonal T cell expansions, eosinophilia, and elevated IgE. Isolated PCP Infection in Children-A Systemic or Specific Immunodeficiency? 1 1 Department of Clinical Immunology, Southampton General Hospital, Southampton, United Kingdom; 2 Department of Child Health, Southampton General Hospital, Southampton, United Kingdom; 3 Molecular Immunology Unit, Institute of Child Health, London, United Kingdom.We report 3 cases of pneumocystis cainii (PCP) pneumonia occurring in Caucasian male infants in the absence of other typical clinical or laboratory features of immune incompetence.Case 1 A 5 month old male infant presented with a short history of poor feeding and respiratory distress. Following a CXR which was characteristic of PCP a brochoalveolar lavage (BAL) was undertaken which confirmed the presence of PCP. The child responded to high dose steroids, Septrin and IVIG and was discharged on day 24. Immunological investigations on admission were as follows: CD3 = 0.23 (2.3-6.5), CD4 = 0.19 (1.5-5) , CD8 = 0.05 (0.5-1.6) CD19 = 0.69 (0.6-3) and NK = 0.07 (0.1-3) . Currently these are; CD3 = 0.27, CD4 = 0.20, CD8 = 0.07, CD19 = 0.99 and NK = 0.29.The stimulation index following PHA was 156. Lymphocytes were low on admission at 1.7 (Normal = 6-9). CD40 ligand was expressed upon activated T cells and CD132 (common g chain) was demonstrated by protein blotting. TCR ah immunophenotyping was normal. He is currently well on prophylactic Septrin and SCIG.Case 2 A 4 month old infant was admitted with a 3 day history of poor feeding and respiratory distress. Immunological investigations on admission were as follows: CD3-2.18, CD4 = 1.5, CD8 = 0.44, CD19 0.99 and NK = 0.24The stimulation index following PHA was 2 on admission and 50 at his most recent review. Immunoglobulins were normal. CD40 ligand was expressed upon activated T cells and protein blotting confirmed the presence of CD132. TCR ah immunophenotyping was normal. Interestingly, this childTs mother had been previously diagnosed with hyper IgE syndrome but he has not yet been shown to have a raised IgE or other typical features of his motherTs condition. The child has had no further clinical episodes associated with immune incompetence and remains well on Septrin and SCIG.Case 3 A 3 month old infant presented with a respiratory distress, weight loss and an enterocolitis. A CXR showed a bilateral hilar infiltrate and a BAL sample confirmed PCP. Immunological investigations on admission were as follows: CD3 = 4.79, CD4 = 4.07, CD8 = 0.61, CD19 = 0.39 and NK = 0.22.The stimulation index following PHA was normal. Immunoglobulins were normal. TCR ah immunophenotyping was normal. CD40 ligand was expressed upon activated T cells.All 3 children demonstrated a susceptibility to PCP without other typical features of immunodeficiency that usually accompany such a presentation in children with SCID or HIGM syndromes. The long term management of such children with apparent single pathogen sensitivity is of interest with regard to the use of a watch and wait approach with imperfect prophylactic treatments or bone marrow transplantation (BMT). 1 1 Central Laboratory for Clinical Immunology, University Hospital Alexandrovska, Sofia, Bulgaria.The bare lymphocyte syndrome is a member of the relatively heterogeneous class of severe combined immunodeficiency and is associated with lack of expression of HLA antigens on some cells of hematopoietic origin. We report cases with BLS who had protracted pneumonia, repeated sever infections of the upper and lower respiratory tract, persistent diarrhea, candidiasis for several months. Peripheral blood immunophenotyping showed prominent decrease of CD4+ T-cells with inverted CD4/CD8 ratio. Antigen-induced lymphocyte proliferation and cell-mediated lymphocytotoxicity were absent in vitro. In contrast the expression of MHC class I molecules was conserved. SBT typing did not show any unknown polymorphisms in HLA-DRB1, DQB1 and DPB1 loci. Since the genes coding for class II polypeptides seemed to be unaffected, the genetic defect in these patients must have concerned the regulation of the expression of the HLA-DR genes. By studding mutations in transacting genes MHC2TA, RFX5, RFXAP we were able to detect genetic defects responsible for a failure to express class II genes in our patients. INTRODUCTION: Intravenous infusions of immunoglobulin (IVIG) every 2-6 weeks have been the standard therapy for patients with primary immunodeficiency diseases (PID). Complications of IVIG therapy limit the use of IGIV at home. Weekly self-administered subcutaneous immunoglobulin (SCIG) infusions at home are becoming an alternative therapy regime. We evaluated a 16% pasteurized, preservative free, liquid, human IgG preparation intended for subcutaneous use with regard to safety and efficacy.METHODS: In two prospective studies, one in the US and Canada (NA study) and one in Europe and Brazil (EU study), 125 PID patients between 3 and 74 years of age self-infused SCIG (VivaglobinR, ZLB Behring) on a weekly basis at home. The patients began the SCIG therapy one week after their last IVIG infusions, and entered a 3-month wash in/wash out period followed by a 12-months efficacy period in the NA study and a 6-months efficacy period in the EU study. Clinical endpoints included the rate of serious bacterial infections (SBI), rates of all types of infections, as well as serum (S) IgG levels observed during the study. Safety variables comprised local and systemic reactions, laboratory investigations and vital signs.RESULTS: A total of 5,953 infusions were administered to 125 patients in the course of the two studies. The patients received a weekly man dose of 158 mg/kg in the NA study and 89 mg/kg in the EU study during the efficacy phase of the studies. Only three SBIs (pneumonias) were reported during the efficacy phase in the two studies; two in the NA study and one in the EU study, resulting in an identical annualized rate of 0.04 SBI per patient. Upper respirator infections were the most frequently reported types of infection. No study drug related serious adverse events were reported in any of the studies. Local injection site reactions, of mostly mild or moderate intensity, dropped rapidly in both studies from initially 85% to about 40% during the course of the NA study and from 65% to about 20% in the EU study.CONCLUSIONS: Two major clinical trials have demonstrated that weekly self-administration of SCIG with a 16% IgG preparation is safe and effective in patients with PID, resulting in normalized stable S-IgG levels and providing satisfactory protection against severe bacterial infections. However, there is a difference in frequency between the Caucasian and Asian populations (approximately 1 in 700 Caucasians and 1 in 18500 Japanese being affected). In addition some IgA deficiency individuals have increased susceptibility to upper respiratory tract or gastrointestinal infections. Primary ciliary dyskinesia is a phenotypically and genetically heterogeneous condition in which the primary defect is in the ultrastructure or function of cilia, highly complex organelles that are structurally related to the flagella of sperm and protozoa. Its clinical features include recurrent sinopulmonary infections, subfertility and laterality defects; the latter due to ciliary dysfunction at the embryological node. Then here we describe for the first time a familiar case of association of IgA Deficiency and Ciliary Dyskinesia.Methods: Clinical and Laboratory evaluation of two siblings who presented to our division with a history of bronchiectasis and recurrent infections.Results: We describe 2 male siblings, 35 and 39 years old, which started to present recurrent infections in childhood, mainly sinus and lung infections. They evoluted with progressive loss of pulmonary function, and one of them had already been submitted to pulmonary transplantation. Family history included a father and older brother with similar symptoms who died because of disseminated bronchiectasis. Laboratory findings showed IgA deficiency with normal IgG and IgG subclasses and normal response to polyssacharide antigens. Further evaluation showed oligospermia with reduction of sperm motility. Atypical Mycobacterial Infection and Chronic Granulomatous Disease: Experience of One Center. Increased incidence of tuberculosis has been reported in CGD patients who live in endemic countries. There are multiple reports of complications following Bacillus Calmette-Guerin (BCG) vaccination in affected patients, manifesting with axillary lymphadenitis and local ulceration. Also known are several cases of disseminated BCG infection in the setting of CGD. Less commonly, atypical mycobacteria other than the vaccine strain, have been recognized as causes of pulmonary infection in CGD.We describe and discuss three CGD patients from one center followed between 1975 and 2004, who have been diagnosed with atypical mycobacterial infection. Patient 1: A 27 year old man with a family history of autosomal recessive CGD, was diagnosed with CGD following Myobacterium fortuitum pneumonia (previously reported). Patient 2: A 7 year old boy, one of three affected siblings with p47phox deficiency, was referred for Myobacterium avium pneumonia and bronchiectasis, but had had numerous bacterial pneumonias previously. Patient 3: A p22phox deficient 7 year old boy was diagnosed with CGD shortly before presentation with disseminated Myobacterium chelonae infection. All three patients responded to anti-mycobacterial antibiotics and have not had recurrent mycobacterial disease to date.In our series, atypical mycobacterial infection either roughly coincided with or preceded the diagnosis of CGD. Surprisingly, all of our patients had recessive forms of CGD, 2 with p47phox deficiency, and one with the rarer p22phox deficiency, despite the fact that recessive CGD is less common and generally thought to be less severe than X-linked gp91phox deficiency. None of the patients had been on interferon gamma (IFNg) or antibacterial prophylaxis at the time of development of their nontuberculous infection. Unlike patients with severe defects in the interferon gamma receptor, none of these patients had recurrences of mycobacterial disease. Although limited in-vitro data have demonstrated no difference in the inhibition of intracellular mycobacterial growth within PMNs and monocytes derived from either patients with CGD or normal volunteers, there appears to be a predisposition to infection with atypical mycobacteria based on our experience. We suggest ruling out CGD in patients with pulmonary or disseminated atypical mycobacterial disease and considering mycobacteria as possible pathogens in infections in CGD patients Su2.51. H. Lin, 1 R. L. Roberts. 1 1 Department of Pediatric Allergy, Immunology, Rheumatology, University of California Los Angeles, Los Angeles, CA, USA.We describe a 19 year old man with a diagnosis of common variable immunodeficiency (CVID) and a two year history of chronic warts. At eighteen years of age, he was diagnosed with CVID following a history of chronic sinusitis, chronic otitis media, and persistent cervical lymphadenopathy. His serum IgG levels were very low (37 mg/dL) although B and T cell subsets were within normal limits. Monthly infusions of intravenous immunoglobulin and continuous oral antibiotics led to improvement of infectious episodes and decreased lymphadenopathy, but warts on all his fingers and elbows persisted and became very disfiguring. He attempted laser therapy, cryotherapy, and imiquimod for removal of warts but met with little success. Difficult-totreat warts are common in patients with immune deficiencies. Most cases are described in patients with cell-mediated immunodeficiencies, and immunomodulatory treatment is often focused on enhancing lymphocyte function. This patient had normal delayed hypersensitivity responses but topical immunotherapy using dinitrochlorobenzene (DNCB) was also not effective. One syndrome, called WHIM (Warts, Hypogammaglobulinemia, recurrent bacterial Infections, Myelokathexis) is characterized by low serum gammaglobulins, failure of leukocytes to leave the bone marrow, and chronic neutropenia. Our patient did not suffer from chronic neutropenia. His therapy for CVID changed from monthly infusions of immunoglobulin to weekly subcutaneous immunoglobulin infusions. Two months into subcutaneous immunoglobulin therapy, his warts resolved completely with no scarring, even though the patient did not receive any specific therapy for the warts while on subcutaneous immunoglobulin. 2 1 Department of Pediatric Allergy, Immunology and Rheumatology, Chang Gung University and ChildrenTs Hospital, Kwei-Shan, Taoyuan, Taiwan; 2 Department of Microbiology and Immunology, Graduate Institute of Basic Medical Sciences, Chang Gung University, Kwei-Shan, Taoyuan, Taiwan.Recent advances in immunologic techniques have lead to increased recognition of primary immunodeficiencies (PID). A review of pediatric patients with suspected immunodeficiencies in Taiwan from Jan. 1985 to Jan. 2005 and molecular/genetic analyses done on some patients were investigated. Based on the International Classification of Disease, Ninth Revision (ICD-9) and published articles, 99 patients with PID (22 females and 77 males) were identified: 59 (60%) with antibody production deficiencies, 15 (15%) with defective phagocyte function, 8 (8%) with combined B and T cell immunodeficiencies, 16 (16%) with T cell deficiencies, and one (1%) with primary complement deficiencies. Those with secondary immunodeficiencies were excluded from the study. Recurrent sinopulmonary infections (62%) were the most common clinical manifestation, followed by sepsis (57%), severe skin infection (40%), splenomagaly/hepatomegaly (27%), central nervous system dysfunction (22%), chronic diarrhea (22%), and failure to thrive (19%). Ten (10%) patients died, seven of infections, one of disseminated intravascular coagulopathy, one of hepatocellular carcinoma and one of lymphoma. Six novel mutations were found from 22 selected patients. Experimental Study of the Complex Bi-Directional Interactions between Human Immunodeficiency Virus Type 1 (HIV-1) and the Protozoan Parasite Leishmania.Chenqi Zhao, 1 Michel J. Tremblay. 2 1 Microbiology-Immunology, Centre de Recherche en Infectiologie, Ste-Foy, QC, Canada; 2 Microbiology-Immunology, Centre de Recherche en Infectiologie, Ste-Foy, QC, Canada.As a result of overlapping geographical distribution, coinfection with human immunodeficiency virus type 1 (HIV-1) and protozoan parasite Leishmania is becoming a common event and presents an extremely serious clinical problem. The two diseases produce cumulative deficiencies of the immune response as both pathogens destroy the same immune cells, exponentially increasing disease severity and consequences. On the other hand, HIV-1 accelerates the dissemination of Leishmania infection and quickens the natural course of the parasitic diseases. The optimal therapeutic approach to HIV/Leishmania co-infected patients is still uncertain, due to the complex pathogenesis of the co-infection and the lack of literature and information. The experiments were performed with physiological experimental models, namely a human lymphoid tissue ex vivo culture system, human primary monocyte-derived macrophages (MDMs), as well as dendritic cells that include human primary monocyte-derived dendritic cells (MDDCs) and a DC-SIGN transfected cell line. The results show that (1) Leishmania enhances HIV-1 replication in both primary human macrophages and in human lymphoid tissues, (2) the Leishmania-directed increase in HIV-1 production is associated with a complex network of proinflammatory cytokines, such as TNF-a, IL-1a, and IL-6, (3) Leishmania also modulates the process of HIV-1 transmission through competition binding to DC-SIGN in dendritic cells, and (4) HIV-1, on the other hand, is also able to promote the intracellular growth of Leishmania in human primary macrophages through an enhanced uptake of the parasite. These findings help to unravel the molecular cellular mechanisms through which the two microorganisms interact, provide novel insight into the complex relationships between both human pathogens, and, most importantly, offer information that may be useful for the design of effective therapeutic strategies to control disease progression in persons dually infected with HIV-1 and Leishmania.Hereditary angioedema (HAE) is an autosomal dominant disease, it manifests as recurrent attaks of intense, massive, localized edema without concomitant pruritus. In case of laryngeal edema, the attack can be life-threatening with the risk of asphyxiation if not treated adequately.AIMS: Our objectives are to study the place of a new intronic mutation (640 GN A) as a molecular basis of HEA typeI, and to investigate the effect of sequence variation within the coding region of C1 inhibitor gene (polymorphism 566 T N G) on disease expression as it has been reported recently (S-A Cumming al, J Med Genet 2003, 40: e114) .METHODS: The studies were performed on 15 members of an Algerian family, five patients with HAE type I and the others are healthy. PCR products were purified using a QIA quick PCR purification kit (QIAGEN, CRAWLEY, UK) and the fragments were then sequenced by direct sequencing of PCR amplified DNA according to the ABI Prism 3700 DNA-Analyser (Biosystems). Fonctionnel tests were perfomed in HepG2 and Hep3B transfected cells by C1inhibitor minigene (all exonic regions and only inton 2 and 3) fellowed by reverse transcription of RNA and agarose electrophoresis of cDNA.RESULTS AND DISCUSSION: Five members of our family have HAE typeI and all of them present a 640 GN A mutation within the intron2 in position+3 (IVS2+3) which isnTt a canonic splice-site region. Fonctionnel tests reveal a presence of 02 different bands, the first one migrates in position 186pb (as W.T band which includes exon2) and the second one in 107pb(as 638 GN A mutation which causes total splicing defect of exon 2). We conclued that our new mutation 640 GN A, never descibed right now, causes a partial splicing defect of exon2. Neverthless, only 02 patients with HAE have a 566 T N G mutation (inherited from the mother whoTs healthy and already described as a polymorphism). Fonctionnel tests have shown no difference in C1 inhibitor synthesis dependig of the presence or no of 566 T N G mutation, and the severe clinical expression of the disease was observed in one patient without that mutation.CONCLUSION: We describe a new intronic mutation in C1 inhibitor gene associated with HAE: 640 GN A that will cause a partial splicing defect of exon2, and we conclude that clinical severity of HAE in our family cannot be explained by the presence or the absence of a 566 T N G polymorphism.Immuno-dermatology Su2.55 . Vehicle Gel in Combination with Narrow-Band Ultraviolet B Phototherapy for Moderate to Severe Psoriasis Vulgaris. Background: Psoriasis is a chronic, autoimmune dermatological disorder characterized by erythematous, hyperkeratotic plaques. Ultraviolet B (UVB) phototherapy is FDA-approved for psoriasis and acts by inducing apoptosis in lesional T-cells. However, phototherapy is often inconvenient and less effective for thick plaques. Oral retinoids are teratogenic and rarely clear psoriasis as monotherapy. Retinoids thin the epidermis in thick plaques, allowing for better penetration of UVB; since limited UVB penetration through thick plaques inhibits efficacy, combining retinoids with UVB would be expected to yield synergistic improvement of efficacy. Bexarotene is a topical retinoid that is FDA-approved for the treatment of cutaneous T-cell lymphoma and is being clinically evaluated in the treatment of psoriasis. Objective: To determine whether bexarotene gel 1% plus narrow-band UVB (NBUVB) phototherapy is more effective for moderate to severe psoriasis than NBUVB plus vehicle gel. Materials and Methods: This was a single-center, double-blind, vehicle-controlled, bilateral comparison of bexarotene gel 1% vs. placebo, in combination with NBUVB, for moderate to severe psoriasis. Nine subjects were randomly assigned gels to be applied to target lesions on the right and left sides of the body. Subjects received NBUVB 3 times weekly for 8 weeks, beginning 2 weeks after the start of topical treatment. Evaluations were done at week 0 (baseline), then weekly for 8 weeks, starting at the initiation of NBUVB. Efficacy was assessed using target lesion scoring and photography. Results: Bexarotene gel 1% plus NBUVB was significantly more effective than placebo plus NBUVB for moderate to severe psoriasis. The changes in target lesion scores were compared for active drug-and placebo-treated sides. The mean decrease in score from baseline for drug-treated lesions was 67.6% (95% CI 50.9%-84.3%), while that of placebo-treated lesions was 48.2% (95% CI 24.0%-72.5%).Because of the small sample size, the nonparametric Wilcoxon ranksum test was used to analyze differences in score changes between two groups. The score improvement with drug was significantly greater compared to placebo (P = 0.04). Scaling, erythema, and induration were reduced to a greater extent with drug. Adverse events were mild and included rash and skin irritation. Conclusions: Compared to placebo, bexarotene gel 1% appeared to increase the efficacy of NBUVB phototherapy with minimal toxicity. Further studies are warranted which include a larger number of subjects. Atopic eczema (AE) is a chronic inflammatory skin disorder. The pathogenesis of AE is not fully understood, but defects in the immune system and the ruptured skin barrier are of importance. The pH of both lesional and non-lesional skin in patients with AE is higher (pH 6) as compared to healthy skin (pH 5.5). The yeast Malassezia belongs to the normal human skin micro flora and can induce IgE and T cell reactivity in patients with AE. Previously, we have identified several IgE-binding components, including a 67-kDa protein, in M. sympodialis extract. Furthermore, based on amino acid sequencing and screening of IgE-binding clones from a M. sympodialis phage display cDNA library, we could isolate a clone with partial sequence of the above mentioned 67-kDa allergen. We have also shown that the 67-kDa allergen is exposed on the cell surface of M. sympodialis. The aim of this study was to investigate whether the 67-kDa allergen can be released under different pH conditions mimicking those of AE skin and healthy skin. M. sympodialis was cultured in Dixon broth pH 5.5 or 6.1 at 32 8C. Culture supernatants were analysed for the presence of IgE-binding components by immunoblotting using serum from an AE patient with specific IgE to M. sympodialis. The result showed that the release of the 67-kDa allergen was substantially enhanced in the culture supernatants with the higher pH. RACE-PCR, cloning and sequencing were used to find the complete coding sequence of the 67-kDa protein which showed similarity to the glucose oxidase family. This sequence was expressed in Escherichia coli as a 6histidine tagged recombinant protein. The IgE-binding frequency of the recombinant allergen was 59%, to 22 sera from AE patients with serum IgE-antibodies to M. sympodialis, indicating that the 67-kDa protein is a major M. sympodialis allergen. In conclusion, we have cloned a major M. sympodialis allergen which is released to a higher extent at pH 6. The data suggest that the skin barrier in AE patients provides an environment that can enhance the release of allergens from M. sympodialis, which can contribute to the inflammation. Purpose: Skin test to exclude/include infection by Mycobacterium tubercle bacillus had been popular in the good olden days, but now has limited its wide spread significance.Methods: Tuberculin skin testing an evidence of tuberculous infection had been in practice since years as diagnostic tool for epidemiological study of the whole/random population, to judge the degree of control of tuberculosis & exclude/include indications for BCG vaccination. Problems had arisen for its limiting value in patients having imminent tuberculous infection as confirmed by isolation of organisms in the sputum smear/culture, a conclusive chemotherapeutic response or clinical evidence of tuberculous infection. Since 1998 to early2002 tuberculous conditions as under with false negative outcome had been documented.Infection by atypical Mycobacterial infection. Advanced age with tuberculosis. False techniques of testing/ improper storage of tuberculin solution.Overwhelming tuberculosis. *Miliary tuberculosis. Tuberculosis with hyperpyrexia. Tuberculosis with generalized skin xanthoma. In some cases no induration had been observed even to 100TU,despite the active tuberculous infection.Results: In face of its limitations as an ideal diagnostic aid in the detection of tuberculosis, its role had been now a supporting test in the absence of BCG with conclusive evidence on radiological & clinical grounds.Conclusions: The cellular mechanisms responsible for skin test reactivity are related mainly to previously sensitizedCD4+ population of lymphocytes that are attracted to the site of skin test. *Tuberculin test had been negative, reactivity restored during the continuation of chemotherapy.Clinical Implications: Tuberculin test despite short of ideal to conclude the diagnosis of tuberculosis still had been an important place in the diagnostic tool list.MIM Objective: We investigated if LNs are required for the induction of protective immunity against Leishmania (L.) major infection.Materials and Methods: We utilized mice lacking peripheral LNs due to in utero blockade of membrane lymphotoxin (LT)(peripheral (p)LN null). Mesenteric LNs were present in pLN null mice. In utero antagonism of membrane LT is transient and the organogenic defects in LN development are irreversible. The secondary lymphoid architecture in the adult progeny of mice undergoing gestational treatment is intact.We also investigated the course of leishmaniasis in mice with genetic deletion of LT ligands (LT-hÀ/À) or of the LT-h receptor(R) which lack skin draining LNs (LT-hÀ/À) or all LNs (LT-h-RÀ/À). To distinguish between the role of LT and LNs in anti-L. major immunity, we also infected wild type Y LT-h-RÀ/À bone marrow chimera which express the LT-h receptor on hematopoetic cells. In addition, C57BL/6 wild type mice were treated with neutralizing LT-h-R-IgG during L. major infection. Splenectomy experiments were performed in order to investigate the role of the spleen in resistance against L. major.Results: pLN null mice of the resistant C57BL/6 strain developed systemic infection with increased IL-4 and reduced ginterferon secretion in the presence of mesenteric LNs. Similarly, in gene deficient mice without local draining LNs (LT-hÀ/À) or lacking all LNs (LT-h-receptorÀ/À) leishmaniasis was disseminated and accompanied by increased secretion of IL-4 in CD4 + T cells. The clinical course of infection was similar in LT-h-receptorÀ/À mice and wild type Y LT-h-RÀ/À bone marrow chimera, which similarly secreted more IL-4. Splenectomized C57BL/6 mice cleared the infection at the same rate as sham operated mice.Conclusions: Peripheral LNs are critical for immunity against L. major in a genetically resistant mouse strain. These LNs provide a milieu which stimulates the induction of a Th1-anti-Leishmania response and prevents Th2 immunity. In the absence of pLNs mesenteric LNs and/or the spleen prime for a Th2 response. Interaction between LT ligands and the LT-R is not required for control of L. major in C57BL/6 mice. Their outstanding therapeutic effects, however, are often accompanied by severe and sometimes irreversible side effects. Thus, the goal of GC pharmacological research is the development of new drugs which show a reduced side effect profile while maintaining the antiinflammatory and immunosuppressive properties of classical GCs. GCs affect gene expression either by transactivation or transrepression mechanisms. Therefore, we aimed to identify ligands of the glucocorticoid receptor (GR) that preferentially induce transrepression while avoiding or at least strongly reducing transactivation. Here we describe a selected non-steroidal selective GR-agonist (SEGRA), ZK 216348, which shows a significant dissociation of transrepression and transactivation activities both in vitro and in vivo. In a murine model of skin inflammation ZK 216348 is anti-inflammatorily active comparable to prednisolone after both systemic and topical application. A remarkable superior side effect profile was found with regard to blood glucose induction, spleen involution and to a lesser extend skin atrophy but not to ACTH suppression. Accordingly ZK 216348 should have a lower risk e.g. Moreover, they are attractive tool compounds for further investigating the mechanisms of GR-action. Objectives: Hypertrophy scar and keloid may be ocurred after burning. Mast cells have important roles in pathogensis of them. The purpose of present study was determination of number changes of mast cells in a experimental model of third degree burn.Methods: A third degree burn was made in 24 rats by direct contact of skin with boiling water for 8 seconds. Rats were divided randomly into four groups. Group1-Burns of control, 2, 3-Burns of these groups were received topical application of unboiled commercial honey one-time per day and twice daily. Group 4-Burns of this group were received topical application of nitrofurazone cream daily. Samples were extracted from 3 rats at day 15 and of another 3 rats at day 30, for light microscopical study were stained with toluidine blue. Numbers of mast cells were counted and Data were analyzed by non-parametric tests.Results: Group 4 had highest number of mast cells at day 15 (30.43 F 41.1) and of at day 30 (31.52 F 41.1). Control group had lowest number of mast cells at day 15 (11.9 F 15.43). Group 2 had lowest number of mast cells at day 30(17.19 F 22.85).Conclusions: It is concluded that topical applications of honey on 3 degree burns, didnTt have significant effect on the number of mast cells in comparison with control and routine treatment groups. We pioneered anticytokine therapy (AT), proposing removal of hyperproduced IFN (Skurkovich, Nature, 1974) and TNF-a together with certain IFNs (Skurkovich, J IFN Research, 1989) to treat various autoimmune diseases (AD). Cytokines induced by IFNgamma (IFN-g), such as TNF-a, IL-1 and their receptors, often work similarly. We had good results treating Th-1 AD, e.g., RA, MS, and corneal transplant rejection with anti-IFN-g. Here we treated Th-1 skin diseases having Th1 cytokines or autoreactive T cells that induce IFN-g and other Th1 cytokines in the lesions. Propionibacteria and estrogen in adolescence, pregnancy and premenstrual period are IFN-g inducers. By day 2 after treatment, most pustular elements had dried. By day 4, infiltrated elements remained but had paled. antigen-reactive T cells and IFNg are found in skin lesions of psoriasis. had a rapid reduction of the erythema leading to disappearance of papular infiltrates and after 2-3 weeks, clearing of plaques and complete remission in most pts. Some with small infiltrates on the legs were given UV treatments, after which they went into remission or completed therapy with 80% plaque reduction. Th-1 cytokines are found in the synovial fluid. Seborrheic dermatitis. Expression of IFN-g mRNA has been detected in skin biopsies. After treatment, itchiness and peeling of skin decreased, and the skin turned paler. Herpes simplex virus type 1 (HSV1). Autoreactive T cells in lesional skin induce IFN-g. 3 to 3.5 hours after treatment, pts. After 2 days, scabs formed, which faded in 5 days. Dystrophic epidermolysis bullosa (a genetic disease but with some AD features). After the 2nd injection of anti-IFN-g, temperature normalized. On day 2, pain, swelling and hyperemia of the ulcerative lesions at the neck disappeared as did signs of infectious damage to the skin on the back. By day 5, erosive lesions on the mouth epithelialized. By day 7, active epithelialization of the ulcerative skin lesions was observed. Dysregulated production of IFNg may exert a pathological effect by increasing virus replication. Within hours after the 1st topical application, itching and pain disappeared. By day 3-4, the eroded area epithelialized. Conclusions: These diseases are connected with immune disturbances in which IFN-g plays a key role and could also be treated, besides with anti-IFN-g, with anti-TNF-a and anti-IL-1 alone or together and soluble receptors to IFN-g, TNF-a, or IL-1, an IL-1 receptor antagonist or anti-CD20. Anti-IFN-g may have fewer complications than anti-TNF-a, such as infection, SLE, demyelination, and others. BuschkeTs Scleredema: Atypical Onset and Evolution. 1 1 Immunology, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj Napoca, Cluj, Romania.In August 2004, a 58-year-old female patient presented to the Emergency Department of the Clinic for significant facial edema and erythema, generalized pruritus, dyspnea, altered general condition. The manifestations appeared after a diet rich in fats and spices. The case was interpreted as QuinckeTs edema and the patient was administered intravenous and oral cortisone and antihistamine preparations. During hospitalization, laboratory investigations were performed, which showed an extremely low ESR (2) (3) (4) (5) , along with leukocytosis that reached shortly 48,500/mm 3 . The patient developed during this interval BuschkeTs scleredema located in the cephalic extremity and upper thorax, which was confirmed by both histopathological examination and antitopoisomerase I antibodies. Ten days after admission, the patient became confused, presented severe headache, impaired vision and temporospatial disorientation. The patient was transferred to the Clinic of Dermatology with the suspicion of tertiary syphilis (in spite of a negative VDRL test), and penicillin treatment was initiated. After 3 weeks, the patient was discharged in an improved condition, but with manifestations of spastic paraparesis. The case is unusual in terms of onset and evolution and no direct causal connection can be established between QuinckeTs edema, BuschkeTs scleredema and spastic paraparesis manifestations. Chronic periodontitis is the result of the immune response to specific bacterial infections in relation to oral flora. The present study investigates the quantitative and qualitative aspects of immune cells in the oral mucosa with correlation to disease progression.Methods. We investigated 30 patients (23-58 years) with chronic adult periodontitis. The diagnosis was verified by traditional clinical and radiologic examinations. The control group was 6 healthy individuals without symptoms of periodontal disease.Informed consent was obtained and biopsies of periodontal tissues were taken after approval from the Ethics committee. Serial frozen sections of gingival mucosa were assessed using the avidin-biotineperoxidase technique with monoclonal antibodies against HLA-DR, CD3, CD4, CD8, TCR gy-chains and CD20. All data were assessed using non-parametric statistics.Results. This investigation demonstrated an interaction between antigens and immune cells which moved from the epithelium to the lamina propria related to the severity of the periodontitis. This process paralleled the loss of the protective potential of the epithelium.Specific immunological features for the early stage of periodontitis include an increase in antigen pressure related to an increase in antigen-uptake by dendritic cells and subsequent persistent inflammation in the epithelium. Further immunological events occur in the lamina propria, with cytotoxic response reduced. The intensity of inflammation in this site was promoted by autoantigens inducing T cell sensitization. CD3+ and CD8+ T cells were the major cell populations associated with tissue damage, while B cells (CD20+) were the most significant cells in the progression of chronic adult periodontitis.Conclusion. Chronic adult periodontitis caused by pathogenic microorganisms may in part be related to host response in an immune-mediated event. Therapy of severe chronic periodontitis should target not only microorganisms which should be eradicated but also the immune response in oral mucosa which may enhance disease progression.Su2.65. Acquired Angioedema and Coagulopathy Several Years after Syphilis. Angioedema (AE) is a rare condition, which is characterized by recurrent, episodic, nonpruritic, subcutaneous or submucosal swelling that primarily affects the face, extremities, upper airways and gastrointestinal tract. AE results from unrestrained activation of the complement system due to an inherited or acquired deficiency of C1 inhibitor (C1-Inh). Acquired AE (AAE) manifests in the adulthood, usually after the forth decade with low serum levels of C1, C1q, C2, C4 and C1-INH activity and can exists in two forms. Type I AAE is mostly associated with lymphoproliferative or neoplastic disorders that result in exuberant complement activation, which overwhelms normal C1-Inh reserves by accelerated consumption. Type II AAE is defined by the presence of autoantibodies against the C1-Inh, which interfere with its function, thus allowing unopposed complement activation.We report the case of a 67-year old man with a distant history of syphilis treated with penicillin, who experienced his first episode of facial edema in 4/02 after ingestion of sweet myrrh root that subsided spontaneously. In 11/02 he developed diffuse swelling of his upper extremities and tongue. No triggers could be identified and a minimal work-up at that time was unrevealing. He remained asymptomatic until 1/04 when he experienced two episodes of tongue swelling that were preceded by minor trauma from poorly fitting dentures. He was treated in a local emergency department with diphenhydramine, prednisone, and epinephrine on both occasions with gradual resolution of edema over several days. He was not taking any medications that could cause angioedema, and did not experience concom-itant urticaria or pruritus. Subsequently, his primary care physician discovered a suppressed CH50, and referred him to our clinic for further evaluation and management of recurrent angioedema.On presentation to our office, he was asymptomatic, and had a normal physical exam. He had not had any swelling until after age 65 and no family history of angioedema. He has never had any bleeding tendency or thrombotic events. His PT and PTT were significantly elevated, which did not correct even at 1:9 mixing with pooled normal serum. Factor XI and XII were 19% and 45%, respectively (50-150%). Fibrin split product was 320 (0-10) and D-dimer was beyond the assay range, even after serial dilutions.Our case demonstrates interconnection between the complement and coagulation cascades at several levels. It also indicates that multiple autoantibodies can coexist in the same individual and suggests that syphilis might have been the inciting culprit, which resulted in AAE. This case exemplifies the diagnostic and therapeutic challenges associated with AAE, which will be presented along with a review of the literature. Atopic dermatitis (AD) is a common dermatologic condition that is characterized by pruritic and eczematous lesions which can be chronic and persistent. Skin lesions are histologically characterized by infiltrating activated T-cells, but the mechanism of this activation remains unclear. IgE-mediated facilitated antigen presentation by IgE-bearing dendritic cells (DC) to T-cells may be a key event in the pathogenesis of AD.Methods. Punch biopsies were obtained from 9 patients with chronic AD after obtaining signed informed consent with the approval of the local ethics committee. AD was diagnosed according to the criteria defined by Hanifin and Rajka. Human skin biopsies taken from patients undergoing cosmetic surgery (n = 9) served as normal controls. The expression of CD80 (B7-1) and CD86 (D7-2) was demonstrated on CD1a+ epidermal dendritic cells (DC) in AD lesions by immunohistological analysis.Results. Cryosections of inflammatory skin were immunostained to localize the CD80+ and CD86+ cells. CD80+ as well as CD86+ cells were identified in the lesional epidermis and dermis. In the epidermis, cells expressing CD80 and CD86 were found at the suprabasal as well as the basal level, scattered throughout the epidermis in an LC-like distribution pattern, being dendritically shaped and exhibiting a membranous staining pattern, suggestive of DC. In this study, we were able to demonstrate that DC are the major epidermal cell population expressing the costimulatory molecules CD80 and CD86 in situ, with AD patients showing the greatest expression.Conclusion. Costimulatory molecules are an essential factor in the generation of an effective immune response, as failure to deliver costimulatory signals during antigen presentation leads to The standard procedure for diagnosing allergic contact dermatitis is to perform a patch test. Since this has several disadvantages, the development of a new in vitro test system would be of immense value. Gene transcripts that distinguish allergics from non-allergics may have the potential to serve as the molecular basis for such a diagnostic tool.In this study, we use the high-density microarray technology in the identification of differentially expressed genes in allergenstimulated peripheral blood mononuclear cells (PBMC) from chromium-allergic patients versus healthy controls.To qualify for the gene expression study, chromium-allergic patients should show a positive patch test to both CrCl 3 and K 2 Cr 2 O 7 and mononuclear cell cultures established from the patients should have a strong in vitro proliferative response to CrCl 3 assessed with the 3 H-thymidine assay. Non-allergic controls would only be accepted for the study if they had no clinical reactions to both chromium compounds and no cellular in vitro response to CrCl 3 . Using these criteria, 3 out of 6 patients and 3 out of 6 controls were selected for the study.Using an Affymetrix GeneChipR, the gene expression was analysed in PBMC cultures grown with 100 m g/ul CrCl 3 or in media alone for 24 h.A total of 26 genes were differentially expressed by more than 2 fold (P b 0.01) in allergen-activated PBMC from patients compared to controls. 18 of these were upregulated whereas 8 were downregulated. Using real-time PCR, the differential expression was confirmed for three selected genes: CISH, ETS2, CASP8. This was statistically significant (P b 0.01) for CISH and ETS2.The 26 differentially expressed genes identified in this study may potentially function as diagnostic markers for contact sensitivity. The long-term use of topical calcineurin inhibitors and corticosteroids raises concerns about immunosuppression and malignancy. Nuclear Factor kappa B (NFkB) transcription factor plays a central role in the progression and maintenance of chronic skin inflammation. This study explores the possibility of using topical NFkB decoy (NFkBD) as a safer therapeutic alternative for skin inflammation. A dustmite antigen induced atopic dermatitis (AD) Nc/Nga mouse model, was employed to further evaluate the potency of NFkBD with approved drugs including corticosteroids, tacrolimus and pimecrolimus. Nuclear localization of the NFKBD was evident throughout the epidermis as well as the dermal layers. Topical NFKBD was more efficacious at both doses (0.25% and 1.0%) compared to topical calcineurin inhibitors, tacrolimus and pimecrolimus. NFkBD therapy, resembling betamethasone, decreased expression of the pro-inflammatory cytokines IL-1beta, IL-6, TNF-alpha and TSLP in inflamed ears. In addition, topical application of NFKBD decreased inflammation, epidermal hyperproliferation and cellular infiltration. Cessation of 0.1% topical betamethasone resulted in a severe rebound of inflammation, whereas NFkBD efficacy was maintained for at least 15 days after treatment termination. Notably, unlike betamethasone that induces skin atrophy within 4 days, prolonged NFkBD application fails to show any such side effect. Conclusion: These results show antiinflammatory steroid-like efficacy of topical NFkBD in skin inflammation without the side effects of topical steroids. Hence, these observations illustrate the potential of topical NFkBD as a novel effective and safe therapeutic agent of inflammatory skin diseases.Su2.71. Dual Diagnosis of Pemphigus Vulgaris and Connective Tissue Disease.Mohsin Malik, 1 A. Razzaque Ahmed. 1 1 Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, MA, USA.Background: Patients with a dual diagnosis of pemphigus vulgaris (PV) and autoimmune connective tissue disease (CTD) have previously been reported. These few reports lack long-term follow-up and clinical details on the relationship of the two diseases.Objective: We report thirteen patients diagnosed with PV who had an additional CTD diagnosis of systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), or both.Methods: We conducted a retrospective analysis of the clinical profile, serological data, treatment, and follow-up of patients seen at one tertiary academic referral center.Results: The thirteen patients were Caucasian with a mean age of onset of PV of 47 years (range 23-71). Ten were female and three were male. In three patients both diseases occurred simultaneously and in the remaining ten PV preceded SLE/MCTD. PV was severe and difficult to treat in twelve patients, though it eventually responded to therapy and these patients were in remission or stable and controlled on tapering therapy. Long-term follow-up, mean 8 years (range 3-18 years), revealed that in six patients the CTD was stable and under control, with periodic need for symptomatic therapy. In seven patients the CTD was controlled but required corticosteroids or other systemic agents. Life-threatening systemic involvement observed in SLE or MCTD, such as renal, cardiac, and neurological manifestations, were absent.Conclusion: The thirteen patients reported here may have a genetic predisposition to develop autoantibodies. Unknown triggering factors are likely to influence the levels of autoantiobodies in such susceptible individuals and result in clinical disease presentation. The differences in response to therapy of the two diseases, PV and CTD, would suggest that both similar and different mechanisms influence autoimmunity, define the extent of disease, and regulate the immune response. Pemphigus vulgaris is a potentially fatal autoimmune mucocutaneous disease associated with production of IgG autoantibodies to desmoglein 3, a 130 kDa epidermal protein. To further characterize the epitope(s) of pemphigus vulgaris antigen we established a human-human hybridoma by fusion of the peripheral blood mononuclear cells with a human and mouse heterohybridoma. This hybridoma designated as PVMAB706 and stable in culture and demonstrated yield of monoclonal antibodies specific for pemphigus vulgaris. Immunofluorescence, immunoblot, ELISA assays demonstrated that the monoclonal antibody bind to the intercellular cement substance and to 130 kDa protein present in the skin and specifically binds to recombinant desmoglein 3 protein, but no to desmoglein 1 protein. The relevance and value of these monoclonal antibodies in the pathogenesis fo pemphigus vulgaris is discussed. Oral pemphigoid (OP) is a chronic autoimmune disease characterized by blisters and erosive lesions in the oral mucosa. We identified an epitope for OP Abs within the integrin alpha (a)6 subunit, and designated four subunit fragments (A,B,C,D). Immunofluorescence studies demonstrated that all the fragments were present in the oral mucosa. Sera of 20 patients with active OP bound only to fragment A and its subfragment A2. The peptide A2.1, within fragment A2, accounted for the binding of all the test sera. Controls were sera samples from 10 healthy volunteers and 30 patients with other pemphigoid diseases. The OP patient sera and immunoaffinity-purified OP sera, rabbit antisera for fragments A and A2, and mAb GoH3 produced basement membrane separation of oral mucosa. Biopsies of oral lesions from OP patients showed that Ab to integrin a6 binds to the roof of the blister and that laminin 5 binds to the base. This in vitro study identifies a peptide in the integrin a6 molecule to which Abs in the sera of OP patients bind, and which may play an important role in the pathogenesis of OP. 1 1 Anatomy, Shaheed Beheshti University of Medical Sciences, Tehran, Tehran, Islamic Republic of Iran.Objective: With regarding acceleration of fractures healing by application of microcurrent electrical stimulation, in this study the effects of microcurrent (microampere) on the full thickness incisional wound healing of rabbits were studied.Methods: 30 male adult rabbits were randomly divided into control and experimental groups. Under general anesthesia and sterile conditions, one full thickness incision on skin of each rabbit was made. From day of surgery experimental group received electrotherapy daily for 2 hours (current intensity: 200 A/cm2, current density: 66 A/cm2, frequency 0.5 Hz). At the end, rabbits were killed by choloroform and 2 samples were obtained from wound tissue and adjacent normal skin for histological and tensiometerical studies. Number of neurtrophils and fibroblasts and cross sections of vessels were counted. Data were analyzed by student T test.Results: Number of fibroblasts of experimental group at seventh day (862.6 + 70.1were higher significantly (P b 0.01) than relevant control group (468.2 + 59). Tensile strength of experimental group at fifteenth day (2138.2 + 212) was higher significantly than relevant control group (1443. 1 + 218.8) .Discussion: It seems the administration of microcurrent serves to boost the electromotive force behind the moving ions and radicals sufficient to allow entery into injured region so that favorable metabolism and repair can take place. Microcurrent by increasing ATP production could accelerate wound healing process.Key words: electrical microcurrent stimulation, wound healing, histology, tensiometery, rabbit. Objectives: The therapeutic effects of Nitrofurazone ointment on healing of infectous and non-infectous second degree burns of rats were studied from morphometric and tensiometric and microbiological examinations point of views.Methods: The type of investigation was experimental. Rats burned according to the standard method and 10% of total body surface of them contacted with boiling water (958C) or 6 seconds. The day of burning was day zero. Half of burns were contaminated by a standard sample of pseudomonas aeruginosa. The burns of Nitrofurazone groups received Nitrofurazone ointment topically one time per day. At 15 th and 30 th day morphometric and microbiological examination and at 30 the day tensiometrical test performed. Data were analyzed by student test method.Results: Results of the Nitrofurazone groups were significantly better than control groups. With regard of the results of present investigation it is concluded that topical application of Nitrofurazone had positive and significant effect on second degree burn wound healing of rats and in non-infectous burns itTs effect was better that in infectous burns. Objectives: The effect of low power Gallium Aluminum Arsenide Laser (Ga. Al. Laser) radiation on numbers and degranulation of mast cells of open skin wound bed of rats from quantitative histological point of view were studied.Methods: 46 male rats were randomly divided into experimental and control groups. Under general anesthesia and sterile conditions one full thickness skin circular wound were made on the dorsum of neck of each rat. The wounding day was day zero. From day one 1/2 doses of anesthetized drugs were injected to all rats and also experimental rats were received Ga. Al. laser which its energy density was Objective: To determine the activation of antigen in red blood cell innate immune reaction main road.Methods: Cancer cells (5  10 6 /ml) and/or Bacillus calmette-Guerin(BCG 0.1mg) or yeast cells(5  10 8 /ml) were added in whole blood cells 0.2ml (or white blood cells 0.2ml) and fresh plasma 0.3ml (or NS 0.3ml) treated by citric acid, and incubated for 1h at 378 to see results. Main aut-come index: IL-8 (ELASA method).Results: It was found cancer cells. BCG and yeast cells can activate hemaimmune reaction, but these antigen not can activate white blood cells immune reaction in not adding plasma group, activation Index (2.124 F 0.860) of IL-8 in antigen adding whole blood cells and plasma group was significantly higher than that (0.390 F 0.080) in antigen adding white blood cells and plasma group (P b 0.01).Conclusion: These results indicate that there is red blood cells main road map of hemaimmune reaction, The red blood cell and complement plays a vital role in hemaimmune reaction road map. Background: With the development of biological drugs it is important to perform immunological studies on patient samples, both to monitor the treatment, but also to increase our understanding of the mechanism of the drug. Thus, both for comparative analyses between samples from different laboratory sites but also for practical reasons in the laboratory, analyses of frozen cells is an option that should be more used. However, the freezing of cells is known to affect their survival and function, and selective loss of certain populations is at risk. This could significantly affect the outcome of various analyses, as compared to the usage of fresh cells. Objectives: To evaluate the effect of the data suggest that the screening ELISA borderline range could be increased and that we could eliminate specificity ELISA testing of borderline positive samples without missing a significant number of specificity ELISA positive samples. In our laboratory, specificity ELISA positive samples with b100 ENA Units were less likely to demonstrate ID reactivity than were samples with N100 ENA Units indicating that only relatively strong ELISA reactivity predicts positive ID results. This analysis has provided a link between ELISA and ID results facilitating interpretation of ENA ELISA results, particularly for weakly positive samples. Antibody arrays, in which antibodies are attached to a surface, are potentially useful tools for a variety of experimental questions. Inherent difficulties in this approach, however, have limited their efficacy. The oligonucleotide tag is a distinctive 30-mer sequence downstream of a common T7 promoter. The antibody binds the protein of interest and indirectly indicates the concentration of analyte. After unbound material is washed away, T7 RNA polymerase amplifies labeled RNA transcripts from the template. In order to interpret the profile, the labeled probe is then hybridized to an oligonucleotide microarray that contains the complementary 30-mer sequences. There are several advantages to this approach, including a greater degree of multiplexing capability than is possible with fluorescent tags, linear amplification of signal, and sensitive and specific detection of protein analytes. Further, this approach is potentially compatible with many experimental designs, including multiplexed analysis of cell-surface markers or lysate preparations of rare cell populations. treated with 0.01% trypsin for 30 min, the Nagalase activity of each fraction increased significantly, suggesting that the Nagalase activity resides on an outer structural envelope protein of the influenza virion and is expressed by a proteolytic process. After disruption of influenza virions with sodium deoxycholate, fractionation of the envelope proteins with mannose specific lectin affinity column along with electrophoretic analysis of the Nagalase peak fraction revealed that Nagalase is the intrinsic component of the hemagglutinin (HA). Similar results were also found in HIV (Yamamoto et al. AIDS Res Hum Retrovirus 11: 1373 and other enveloped viruses (e.g., Sendai, rubella and measles viruses). INTRODUCTION Although nitric oxide (NO) is a major regulator of inflammation, little attention has been focused on upstream regulators of NO such as the solute carrier (SLC) family of molecules. SLC7A2 supplies L-arginine to the inducible NO synthase (iNOS) and is required for sustained NO production by macrophages. Bacterial lipopolysaccaharide (LPS) and the cytokine interferon-g (IFN-g) are strong inducers of iNOS in macrophages. OBJECTIVES Identify immune cytokines modulated in LPS and IFN-g stimulated SLC7A2 -/macrophages and examine T-dependent immune responses in SLC7A2 deficient mice. MATERIAL AND METHODS Adult wild type, iNOS deficient and SLC7A2 deficient mice of both sexes in the C57Bl/6 strain were stimulated with thioglycolate for 72 h. Peritoneal macrophages (PM) were primed with 20 units/ml IFN-g in culture media for 2 h, and then 100 ng/ml LPS were added for an additional 17 h. Total RNA was isolated and after reverse transcription, cDNA products were used for amplification of SLC7A2, iNOS and several cytokines by real time PCR. In other studies, mice of the three genotypes were inoculated intraperitoneally with 100 Ag DNP-KLH in FreundTs complete adjuvant on day (d) 0 and boosted on d21 using 100 Ag DNP-KLH in FreundTs incomplete adjuvant. Serum was collected at d0, d7, d14, d21, d28 and d35 and tested by ELISA for immunoglobulin production. IFN-g, IL-10 and IL-4 expression were all significantly increased in iNOS and SLC7A2 deficient PM compared with wild type control PM. IL-6 and TGFg-2 expression was significantly decreased in iNOS -/and SLC7A2 -/-PM compared with wild type controls and TGFg-1, TNF-a and phospholipase A2 were unchanged. Serum IgG2a levels were dramatically reduced in iNOS and SLC7A2 deficient mice during the primary immune response, but returned to wild type levels after the boost. Serum IgM, IgG1 and IgG3 levels were normal in both genotypes. CONCLUSIONS Overall, SLC7A2 deficiency in macrophages results in a similar phenotype to iNOS deficiency. Interestingly, when L-arginine metabolism by iNOS is absent, there is an upregulation of arginase-I expression, presumably to utilize the excess L-arginine. Whether this effect is dependent on SLC7A2 remains to be determined. Despite increased INF-g expression by iNOS and SLC7A2 deficient PM, IgG2a production is reduced following immunization, suggesting that the increase in Th2 cytokines IL-4 and IL-10 dominate this response; so the absence of SLC7A2 and iNOS genes can modulate cytokines levels expression. And defining this function in inflammation; that is, understanding molecular pathways controlling NO production in macrophages, is an important step towards developing improved therapies for inflammatory diseases and primary immunodeficiencies.Su2.95. Improved Immunological Methods Using Peptides: Western Blots, Peptide Arrays, Kinase Assays and ELISAs. Their use, however, in protein arrays is hampered by ineffective and variable binding efficiency of peptides that could result in low sensitivity, false positives and inconsistent signals; in Western blots and ELISAs their use is limited due to their small molecular mass. To overcome these hurdles, we apply inteinmediated protein ligation (IPL) in which a peptide possessing a Nterminal cysteine is linked to the carboxyl terminus of a reactive carrier protein via a peptide bond. The ligation products are then arrayed on to a membrane or used in Western blot analysis. In this poster, we demonstrate multiple applications of intein technology in immunological methods: a) Easy generation of carrier-peptide substrates suitable for Western blot analysis (1) . c) Generation of tailored substrates for kinase and phosphatase assays ( Background: The enzyme-linked immunospot (ELISpot) assay is useful in measuring responses to vaccination and changes following immunotherapy. We have developed, optimized, and validated a customized BBI ELISpot kit for immunogenicity assessments in NIAID-sponsored HIV clinical trials, and compared its precision in fresh and frozen samples collected at one month intervals, while measuring biological variability.Methods: The capture antibody, detection antibody, Streptavidin-HRP, and substrate were titrated for optimal performance of the BBI kit and used according to kit instructions. Repeatability was characterized using PHA and CEF pool (CMV, EBV and Flu peptides) in a minimum of triplicate wells. The within donor variability was determined using 3 different donors with 5 time points collected one month apart. Samples were assayed by ELISpot using fresh cells in real time and by batch assay using frozen cells at the end of 6 months from the first time point. Within donor variability was presented as mean SFC per 200,000 PBMC F SD (%CV) for CEF for each of the 3 donors. PHA SFC were given per 100,000 cells.Results: Repeatability within assay was similar whether CEF or PHA was assayed fresh (13% vs. 16%) or frozen (16% vs. 11%). For PHA, the measurements were 136 F 27 (20%), 184 F 75 (41%), 211 F 18 (9%) for fresh, and 252 F 72 (28%), 351 F 136 (38%), and 329 F 117 (36%) for frozen. The CEF SFC values were higher for donor 1 in fresh vs. frozen; however there was no difference in the other 2 donors. The variability among samples from the same donor was higher in the batched frozen samples than in the individually run fresh samples. PHA showed opposite results, with all fresh samples having lower SFC values than frozen, and the variabilities were similar except for donor 3, which was higher in the frozen samples.Conclusion: Both fresh individual and frozen batch assays demonstrated similar intra-assay repeatability. For CEF the mean values for 2 of 3 donors over 5 time points were essentially the same for fresh and frozen samples. Interestingly, the variability within a donor was greater in the batched frozen samples than the fresh samples. This suggests that day to day variations in freezing procedures may result in greater variability than testing real time on different days with donor samples drawn at different times. HLA-Typed PBMC Samples with Established Antigen/Peptide Reactivity for Accelerating and Standardizing Human Immunological Research. We have developed a protocol to cryopreserve human peripheral blood mononuclear cells (PBMC) while maintaining full functionality. The thawed PBMC display N 80% viability, and when tested for peptide or protein antigen-induced T cell recall responses in cytokine ELISPOT assays, the frequencies and per-cell cytokine productivities of the thawed cells approximate 100% of the fresh PBMC. Since serum is a highly variable breagentQ that affects the results, we developed serum free freezing and testing media towards standardization. The PBMC of each donor has been characterized for reactivity to a panel of 23 individual peptides (common viral Class I-restricted determinants) and 5 protein recall antigens, recognized by CD8 and CD4 cells, respectively. Up to 1,500 vials of each of the characterized samples have been cryopreserved and made available as positive and negative controls for T cell monitoring in ELISPOT, ELISA, cytokine bead array, tetramer/pentamer, and cytokine capture assays. Ready access to such highly characterized PBMC should facilitate human immunological research and offers reference samples for assay standardization within laboratories, and between different laboratories.Su2.98. Cell Proliferation Index. A Reliable and Validated Method That Quantifies Cell Proliferation According to CFSE Dilution. J. C. Crispin, 1 M. I. Vargas-Rojas, 1 J. Alcocer-Varela. 1 1 Department of Immunology and Rheumatology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Mexico City, Mexico.Cell proliferation is a mechanism intimately linked to the immune response. Several cellular activation-induced pathways converge in it, and it takes place synchronously to the acquisition of effector functions and phenotype. Thus, its measurement is essential because it is an important marker of response against a variety of stimuli. Several methods for the quantification of cell proliferation are available. However, it requires the handling of carcinogenic and radioactive materials. Moreover, when more than one cell population is being cultured, the method does not distinguish between them. An alternative approach is based on labeling T cells with carboxyfluorescein diacetate succinimidyl ester (CFSE). CFSE is membrane-permeable fluorescent dye that binds covalently intracellular molecules. When a cell divides, the fluorescence intensity halves in each of the two resultant cells. Thus, each round of cell division produces a population of cells that have one half of the fluorescence intensity than the cells they arose from. Using a FACS, cells that have not proliferated can be easily distinguished from cells that have proliferated, and according to the dilution of the dye, one can assume how many division rounds each cell has gone through. Its principal drawback is that results are semi-quantitative and, when differences are subtle, comparison is difficult. In the present work we present an algorithm that translates the semi-quantitative data obtained from the FACs and yields a numerical result. Materials and Methods. PBMC (from healthy donors) and Jurkat cells were used. Cells were cultured during 72 hours and stimulated with either plate bound aCD3 plus soluble aCD28, or PHA. Cell proliferation was quantified by: a) manual count of live and dead cells (according to trypan blue exclusion); b) [ 3 H]-thymidine uptake; c) Cell Proliferation Index (CPI). For the FACS studies, a histogram (FL1 [CFSE intensity] vs. cell number) was drawn with the target population. A first marker (M1) was set according to a negative control (non-stimulated cells). Next, markers (M2, M3, etc) were set progressively, each one including a daughter population (considering the geometric mean must halve in each population). Hence, the cells accounted within the M1 have not proliferated, the cells in M2 have undergone one round of proliferation, the cells in M3 three rounds, and so on. The results obtained with each method were compared. Cell proliferation was detected by the three methods. The results obtained with the CPI correlated closely to the measurement of cell proliferation with [ 3 H]-thymidine uptake (R = 0.92, P b 0.0001). CPI is a sensitive and reliable means of quantifying cell proliferation. Evaluation of CellPrep, an Automated Cell Washing Instrument for Analysis of Surface Markers on Leukocyte Subpopulations Via Flow Cytometry.J. 1 1 Biomedical Research Division, Beckman Coulter Inc., Miami, FL, USA. There have been significant improvements in reagents, procedures, and instrumentation that have allowed for the introduction of automated techniques for the preparation of cell samples prior to flow cytometric analysis. An invaluable advantage to automation is the standardization and reduced bhands-on timeQ as compared to manual techniques.The current and most commonly used technique of centrifugation to wash cells by removing contaminating materials prior to flow cytometric analysis, does not lend itself well to automation. Additionally, subjecting cell populations to both g-force and intense cell-to-cell contact through centrifugation may alter sensitive activation and signal transduction expression. To simplify and automate cell washing in tubes, the non-centrifugal CellPrep instrument was designed and developed to wash cells using polysulfone hollow fibers.The present study was carried out to compare scatter profiles, surface antigen expression, and recoveries of rare and non-rare events in unwashed, centrifuge-washed, and CellPrep-washed cell samples. It has been shown that the CellPrep instrument can be used successfully to wash peripheral blood cell suspensions with no adverse effects on the phenotypic and scatter profiles, as well as cell recoveries.Su2.100. Optimization of the Aspiration Dose of IL-1ra Preparation To Stop an Inflammation in the Mouse Respiratory Tract.A. 3 1 Protein Biochemistry, Institute of Highly Pure Biopreparations, Saint-Petersburg, Russian Federation; 2 New Drug Formulations, Institute of Highly Pure Biopreparations, Saint-Petersburg, Russian Federation; 3 Immunopharmacology, Institute of Highly Pure Biopreparations, Saint-Petersburg, Russian Federation.Hyperproduction of interleukin-1 (IL-1) is a main factor to provoke inflammation. The interleukin-1 receptor antagonist (IL-1ra) may serve as a promising therapeutic and prophylactic agent for stopping inflammatory processes, including the infectious ones. In this study the prophylactic efficacy of IL-1ra was assessed after aerosol application to mice, in which inflammation was induced by instranasal instillations of LPS. The efficacy of IL-1ra was studied after two routes of application-the aerosol and the injection one. Based on a theory of multifactor analysis, the aspiration dose of IL-1ra and the particle size distribution in aerosol were optimized.It was demonstrated that IL-1ra applied in a form of aerosol caused the decrease of inflammation in the respiratory tract of a mouse. 50% decrease of inflammation was registered for the aspiration dose of IL-1ra 150 mcg/mouse. The same effect for the injection IL-1ra was registered at a dose of 270 mcg/mouse of IL-1ra. The anti-inflammatory effect of the fine-dispersed fraction of IL-1ra (particles of 2 mcm in size) was 30% higher than that of the coarse-dispersed fraction (particles of 10 mcm in size). The resultant data demonstrated the advantage of aerosol route of administration of IL-1ra over the injection one.Su2.101. The Use of Arginine-Rich Peptide Conjugated Antisense Oligomers To Alter Immune Function. Phosphorodiamidate Morpholino Oligomers (PMOs) are effective antisense agents for inhibiting gene expression; however limited uptake into populations of immune cells without the use of mechanical and chemical procedures often detrimental to cellular functions, restricts their usefulness. A strategy shown to enhance cellular uptake of PMOs into fibroblasts is the conjugation of an arginine-rich peptide to the oligomer. To examine uptake of these peptide-conjugated PMOs into lineages of primary T cells, B cells, and NK cells, as well as bone-marrow derived macrophages, and dendritic cells, we used flow cytometry to measure the presence of a fluorescein-linked PMO in treated cells. Uptake of the oligomer into these cell types is greatly enhanced by the addition of the argininerich peptides compared to unmodified PMO. Furthermore, differential uptake into different cell types was observed and found to be dependent on the amino acid composition of the peptide and the activation status of the cell. Demonstration of an antisense-specific effect is shown by targeting the expression of CD45 in cells treated with the PMO peptide conjugate CD45-(RxR)4. Antisense efficacy was also demonstrated by forcing alternative splicing of the CD45 mRNA using antisense PMO conjugates targeting the splice junctions of exons 4, 5, & 6. Successful uptake of PMO peptide conjugates into immune cells and target inhibition of gene expression suggests that PMO modified with arginine-rich peptides could potentially be used as an immune modulating therapeutic strategy.Su2.102. A Multi-Level Approach to Analyzing Immune Responses in Targeted Cells by Combining Cytomic and Proteomic Techniques of Cell Sorting and Protein Fractionation.S. 1 1 Biomedical Research Division, Beckman Coulter Inc., Miami, FL, USA. In order to simplify the study of bSystems BiologyQ and facilitate the transition to bCytomicsQ i.e. linking of genomics and proteomics to functionality of the cell, a multi-level and multi-pronged approach needs to be pursued. This requires that cell-based events that have so far been interrogated in isolation, for e.g. gene expression, protein synthesis, phenotype and function, signal transduction etc. now need to be integrated to better understand the bcomplexQ cellular response. This continuum can to some extent be achieved by integrating cellular analysis techniques (for e.g., flow cytometry, imaging, multiplex assays) with genomics and proteomics techniques to understand the various aspects of such a response, thus accomplishing the unified evaluation of the cell. In the current study, the authors have attempted such an evaluation by integrating well-known techniques of flow cytometry-based phenotypic analysis and cell sorting with protein fractionation and analysis to better understand the complex nature of an immune response.However, the application of these assays for identifying a specific immune response or lack thereof is complicated by the fact that most individuals have different HLA alleles and haplotypes. Furthermore, the many HLA alleles in the population tend to differ functionally. One must realize an HLA type in order to determine which class I or class II MHC molecule presented a particular peptide epitope in a positive ELIspot experiment. There are now 349 HLA-A, 627 HLA-B, 182 HLA-C, 394 DRB1, 80 DRB2-9, 28 DQA1, and 61 DQB1 alleles at the respective loci. Our first HLA resource is to provide researchers with a definitive class I and class II HLA type using DNA sequence-based typing. We have typed samples from populations throughout the world, identifying new and rare HLA alleles and haplotypes in a racially unbiased and precision manner. Of the MHC Class I and Class II linkage disequilibrium haplotype determining loci, we have typed 100 HLA-B and 55 HLA-DRB1 alleles. Our HLA typing laboratory is ASHI/CLIA accredited and is directed by ABHI certified laboratory director. ouhsc.edu) through which we provide an online resource whereby all known HLA class I and class II peptide ligands and motifs are catalogued. The HLA Ligand Database provides a resource whereby scientists can find or predict peptide epitopes that bind to particular HLA molecules. Potassium channels on immune cells have gained attention recently as promising targets of immunotherapy. We therefore turned our attention to potassium channels on antigen-presenting cells, specifically human dendritic cells, whose K+ channel profile has not yet been described in the literature. We generated a population of immature dendritic cells by culturing monocytes from the blood of healthy human donors in vitro with GM-CSF and IL-4, as previously described, and then stimulated these cells with LPS or TNF-a to induce maturation. Whole-cell patch clamp analysis of these cells revealed an inward-rectifying K+ current at early timepoints after stimulation, replaced by a mix of voltagegated Kv1.3 and Kv1.5 channels at later stages of maturation. The identity of these channels was established by characteristic inactivation curves and pharmacological blockade. Further, immunofluorescent staining confirmed the presence of Kv1.3 and Kv1.5 on the surface of stimulated cells, colocalizing with HLA-DR. In order to determine whether these channels have a functional role in DC maturation, we then analyzed DCs stimulated in the presence of pharmacological Kv1.3 blockers, and found that both CD83 and CD80 upregulation and production of IL12 and IL6 were significantly impaired (28% decrease in CD83, 46% in CD80, 16% in IL6, 32% in IL12). When induced, this mutant protein associates with other Kv1 family members in membrane tetramers and blocks channel function. We compared DCs infected with this Kv1 dominant-negative construct to DCs infected with a control virus coding for luciferase, and found that maturation of DCs expressing the Kv1.x viral product was highly reduced (53% less CD83 expression than control). Overall, our data are the first to report Kv1.5 and Kv1.3 expression on mature human DCs, and further indicate that these channels have a functional role in DC maturation. Our results therefore bolster the argument for K+ channel blockade as an immunotherapeutic strategy targeting mature antigen presenting cells, which has implications for the treatment of a wide range of immune-mediated diseases. The therapeutic mechanism of Kv1.3 blockade in EAE, for example, has not yet been fully explored, and may include an effect on antigen presentation and priming of T-cells in conjunction with a direct suppression of T-cell function. Indeed, immunohistochemical staining of plaques in the brains of MS patients reveals Kv1.3 and Kv1.5 on microglia, offering a first piece of evidence that targeting these channels may affect antigen presentation to T-cell infiltrates within the brain. As immune modifying therapies continue to be employed, developed and expanded to treat diseases such as rheumatoid arthritis, inflammatory bowel disease, HIV, HCV and cancer, a need exists to assess their effects on the immune system. CylexR has developed several cell-based assays to measure the functional activity of lymphocytes from a small amount of whole blood. These assays utilize in vitro stimulation of a patientTs blood sample, followed by magnetic isolation of specific sub-sets of lymphocytes. ImmuKnow TM , an FDA-cleared assay, measures the global immunological response of CD4+ cells and the T-cell Memory assay is a research test that measures antigen-specific responses of CD3+ cells. By examining both global and antigen-specific cell mediated immunity, the Cylex platform is useful for monitoring the immune status of patients receiving immune modifying therapies. In this study, immune responses of apparently healthy individuals and drug-induced immunosuppressed transplant recipients to foreign antigens including Influenza, CMV, Tetanus and EBV as well as global immune responses to PHA were measured. Transplant recipients undergoing lymphocyte depleting therapies showed a marked decrease in global and antigen-specific responses. Recovery of these immunological responses occurred gradually over the next 6-12 months and was largely independent of the absolute lymphocyte count. Healthy volunteers with known natural exposure or vaccination showed significant responses to vaccine antigens whereas those not exposed or unvaccinated showed no response. In a study of response to tetanus, 90% of apparently healthy adults had an in vitro response to tetanus toxoid, but only 18% of transplant recipients and 28% of HIV+ individuals had positive antigen responses. By examining both global and antigen-specific immune responses, the Cylex platform is useful for monitoring the effects of immuno-modulating therapies, whether they are designed to enhance or suppress the patientTs immune response. Murine models of human disease are established immunological tools, prompting the need for a product to expand murine T cells ex vivo. The antigen CD28 provides an important T cell costimulatory signal. DynabeadsR coated with anti-CD3 and anti-CD28 monoclonal antibodies (mAbs) were used in short-term (b14 day) cultures of BALB/c CD4+ or CD8+ T cells in vitro, in order to define ratios of the mAbs and of beads:cells allowing optimal upregulation of activation markers, increase in cell volume, and expansion of T cell numbers. The performance of the beads in both short and long-term cultures of various lymphocyte populations was then evaluated. Over 35-fold expansion of BALB/c CD4+ T cells was observed over 18 days; robust proliferation of CD8+ T cells, mononuclear cells and antigen-specific T cell clones could also be achieved, without loss of function. CD4+ T cells could be expanded for periods of z6 weeks; CD8+ T cells could not be expanded for N3 weeks. Following initiation of the cultures, restimulation was necessary at 7-12 day intervals. In summary, we have thus developed and rigorously validated a product allowing the expansion of diverse populations of murine T cells in vitro. Characterization of Endogenously Loaded Rhesus Macaque MHC Class I Peptides. A. R. Gilb, 1 H. D. Hickman-Miller, 1 W. Bardet, 1 A. D. Luis, 1 D. I. Watkins, 2 K. Jackson, 1 W. H. Hildebrand. 1 1 Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA; 2 Wisconsin Regional Primate Center, University of Wisconsin, Madison, WI, USA.The SIV-infected Indian Rhesus Macaque (Macacca mulatta) is an often used animal model for the study of HIV infections in humans. In terms of anti-viral immune responses, both macaques and humans mount strong cytotoxic T lymphocyte (CTL) driven anti-SIV and anti-HIV immune responses, respectively. In order to compare human and macaque anti-viral immune responses, to test SIV vaccine strategies, and to interpret viral escape mutants, human and macaque class I major histocompatibility complex (MHC) peptide binding properties must be elucidated. Our laboratory is focused upon the amino acid sequencing of pooled motifs and of individual peptide epitopes that are endogenously generated, trafficked, loaded, and class I MHC presented. In this study we transfected 4 macaque class I molecules (Mamu A*02, A*11, B*01, and B*12) into a human cell line and harvested 10-20 milligrams of each macaque class I molecule. Eluted peptides were initially subjected to 14 cycles of Edman degradation and the resulting data shows that macaque class I molecules are endogenously loaded with nonamers demonstrating P2, P9, and other ancillary anchors. At least 10 individual ligands were sequenced by MS/MS for each of the macaque class I, demonstrating variability in length as well as variability in sequence as compared to the pooled motifs. Finally, anchors and motifs not detected by other methods are apparent through the characterization of endogenous ligands. In summary, the characterization of endogenous macaque class I peptide epitopes provide a more thorough understanding of immune responses in this animal model. Introduction: Measurement of differential RNA expression in multicenter clinical trials requires special attention to the quality in sample preparation. Sample collection and handling can adversely affect results; therefore, quality metrics are required to detect and assess the potential errors induced by these factors. We compared the reliability of newly devised quality metrics derived from fitted statistical models of probe level data from high-density oligonucleotide microarrays and compared them to standard GeneChipn microarray quality metrics. Method: We devised metrics using the Robust Multichip Analysis (RMA) process for deriving probe set summaries from GeneChipn microarrays. The first metric, Normalized Unscaled Standard Error (NUSE), provides a measure of relative chip quality derived from the residuals from the RMA model. The second metric, the Relative Log Expression (RLE), is an absolute metric that gauges variability of expression measures by summarizing the distribution of relative log expressions within a set of microarrays against a reference set. These metrics were compared to standard quality metrics: Percent Present calls, GAPDH 3V/5V, Background, and Scaling Factor. Two clinical trial sample sets were assessed: 368 microarrays from a Type I diabetes trial and 350 arrays from a ragweed allergy trial. A set of standard normal human control samples were sent blinded within patient sets during the course of ITN clinical trials and were used as the reference set against which RLE assessments were made. Result: NUSE and RLE metrics detected systematic variation within certain participant sets that were not detectable using the standard Affymetrix quality metrics. Elevated GAPDH 3V/5V ratios typically cited as an indicator of poor quality RNA showed no relationship to quality when applying the NUSE and RLE (cor, 0.09). Hybridization/ washing artifacts were easily visualized by plotting NUSE residuals. While not always true, Percent Present calls provided the closest approximation to NUSE and RLE; in cases of extremely low Percent Present, NUSE and RLE are adversely affected (cor, -0.50). In both trials in which these metrics were applied, we identified chips within a participant time series that required exclusion from the analysis that would not have been discovered otherwise. Conclusion: In differentiating NUSE from RLE, NUSE values have no units and can only be used to assess the relative quality of arrays within an analysis set; RLE summaries provide a measure of reproducibility of gene expression data that can be compared across batches, experiments, or trials. Reflecting variability in expression measures, these proposed metrics provide a better basis for judging quality compared to standard metrics. The class I and class II Human leukocyte antigens (HLA) mediate most, if not all, adaptive immune responses. Since each individual has a different combination of class I and class II HLA molecules inherited from her/his parents, the immune response to infection and vaccination differs respectfully from person to person. In addition, many autoimmune diseases, such as arthritis and diabetes, are associated with particular class I and/or class II molecules. Knowledge of a patients/populations HLA molecules therefore becomes a key element in vaccine design and uncovering autoimmune triggering mechanisms. HLA DNA sequence-based typing (SBT) represents a method that identifies all polymorphisms in a racially independent manner. Our laboratory pioneered and continues to employ a precision HLA SBT method for studies of bone marrow transplantation, vaccine development, and autoimmunity. The class I and II HLA SBT process is split into three steps-PCR, DNA sequencing, & data processing-and here we report on the evolution of these 3 steps during the high resolution SBT of more than 20,000 individuals in the last 8 years. We describe the migration of our method from a solid phase sequencing chemistry to a capillary DNA sequencer, we discuss the location of PCR and DNA sequencing primers, we compare SSP and RSCA methods for the resolution of ambiguities, we discuss the software packages available for data processing, and we describe the costs now associated with HLA SBT. Organ Transplantation Su2.112. On the Possibility of Oral Tolerance To Be Used in Graft Transplantation.Xiao-Bin Zheng. 1 Research and Development, Beijing Zhongbangyumin Sci-trade Company Co. Ltd., Beijing, China.Oral tolerance has been studied for many decades. And oral tolerance has been used to treat autoimmune diseases, such as rheumatoid arthritis, uveitis, EAE, as well as some others, experimentally and/or clinically. Interestingly, oral tolerance has also been used in reproductive immunology, i.e. It has been used to induce maternal tolerance to paternal antigens, in order to establish an immune tolerance to the semi-allograft fetus by the mother to treat the abortions. But, as a way of being possible to establish an antigen-specific immune tolerance, why should not we use it to the transplantation immunology, trying to use this way to induce an immune tolerance to the transplant graft antigens, for the purpose to establish an immune tolerance to avoid the graft rejection. Evidence for Naturally Occurring and Induced Regulatory T-Cells in Non-Human Primates. Non-human primates (NHP) are often used as preclinical model for the evaluation of tolerance inducing therapies. Regulatory Tcells (Treg) may be crucial for the maintenance of tolerance. Here we describe the identification and characterisation of Treg in NHP. CD4+CD25+ cells were isolated from peripheral blood mononuclear cells (PBMC) of healthy monkeys and from PBMC of 3 long-term drug free kidney transplant recipients (long-term trans-plant survivors, LTS). One LTS monkey is 23 years post transplantation after treatment with pre-transplant bloodtransfusions and CsA for 1 year and two monkeys are 3 years after cessation of treatment with costimulation blockade and CsA. Several characteristics known to be specific for CD4+CD25+ Treg in humans and rodents were investigated.Naturally occurring CD4+CD25+ cells were present in healthy monkeys as well as in the LTS monkeys. Similar to CD4+CD25+ T-cells in humans, CD4+CD25+ T-cells in NHP do not proliferate upon polyclonal or allogeneic stimulation. However, in contrast to humans, proliferation is only slightly increased upon addition of IL-2.When CD4+CD25-cells are activated by a polyclonal (ConA) or allogeneic stimulus, CD4+CD25+ cells can suppress this proliferation and, although not proliferating themselves, the CD4+CD25+ cells cannot inhibit proliferation when they are irradiated. CD25+ cells have, in accordance with human CD25+ cells more intracellular CD152 than CD25-cells.CD4+CD25+ cells are present in the LTS monkeys in the same numbers as can be found in healthy NHP and they also do not proliferate upon ConA or allogeneic (donor specific and 3rd party) stimulation. To evaluate possible donor specific regulation in other subsets, anti-TGF-beta and anti-IL-10 were added to whole PBMC cultures. In the LTS monkey 23 years post transplantation, regulation seems to be TGF-beta mediated, which correlates with the presence of large amounts of latent TGF-beta in the kidney. In the other two LTS monkeys, neither anti-TGF-beta nor anti-IL10 seems to uncover proliferation, but the combination of both antibodies induces increased proliferation against the donor.Naturally occurring Tregs, with similar characteristics as described in humans are present in NHP and can suppress CD4+CD25-cells. Although CD4+CD25+ cells in LTS monkeys are immunosuppressive, this is not donor specific, and therefore, donor specific regulation may be confined to another T-cell subset. Objectives: Kidney graft recipients with stable renal function in absence of immunosuppressive therapy are characterized by a skewed TCR Vbeta chain usage, essentially in the CD8+ subset. Therefore, the present study analyzes in more detail phenotypical and functional alterations of CD8+ lymphocytes in these drug-free tolerant patients (DF-Tol).Methods: Peripheral blood CD8+ lymphocytes from DF-Tol, chronic rejection (CR), healthy controls (HC), and patients with stable kidney function under immunosuppression (StA) were analysed by flow cytometry for phenotypic and cytotoxic markers. Apoptosis was measured by annexin-V staining and proliferation by CFSE.Results: Phenotyping revealed an increase of CD45RA-CCR7+ central memory and a decrease of CD45RA+CCR7-effector CD8+ lymphocytes in DF-Tol versus CR. The expression of CD28+ and CD27+ on effector and effector memory CD8+ lymphocytes was decreased in CR, with a high correlation between both markers. These profiles were stable over time and independent of treatment. The cytotoxic nature of CD8+ CD28-cells was indicated by the higher expression of perforin and granzyme A in CR versus DF-Tol, the inverse correlation of these markers with CD28 and CD27 expression, and the increased expession of the cytotoxic marker CD57 on the CD8+ CD28-subset. The CD8+ CD28-lymphocytes expressed lower levels of Fas and were less sensitive to apoptosis than their CD8+ CD28+ counterparts. HC displayed the same profile as DF-Tol, indicating an increase of CD8+ CD28-effector lymphocytes in CR rather than a decrease in DF-Tol. Sta displayed a mixed profile, with some patients resembling DF-Tol and others mimicking CR.Conclusion: A strong cytotoxic CD8+ CD28-signature differentiates CR from DF-Tol and HC, suggesting a suppression of pathological cytotoxicity in DF-Tol. Further investigation of the targets of these cytotoxic cells and evaluation of these profiles to identify patients at risk for CR are warranted. While the relevance of pre-formed anti-HLA antibodies is well determined, less known is the role of alloantibodies produced after cadaveric kidney transplantation on graft outcome. The aim of this study was to evaluate the incidence, dynamics and profiles of developed post-transplant anti-HLA antibodies and their impact on graft outcome in kidney recipients. We retrospectively investigated 72 patients with no detectable alloantibodies prior to their first cadaveric kidney transplantation for the period October 1998-January 2004. All patients received triple immunosuppressive therapy. Biopsy-proven acute rejection was observed in 11.1% and chronic rejection-in 15.3% of the recipients. The alloantibody profile was determined with Flow-PRA Screening and Specific Tests (One Lambda, USA). Anti-HLA antibodies after transplantation were detected in 22.2% of the studied patients. Donor-specific reactivity was determined in 75% of the alloantibody positive patients. A correlation between triplet mismatches and alloantibody production was observed. Most of the recipients (81.25%) produce alloantibodies in the early post-transplant period. Alloantibody titre evaluation demonstrated that patients with high titers experienced acute rejections and early graft loss, while in those with low and stable titers chronic rejections were more common. In conclusion our data suggest that recipients with post-transplant HLA-reactive antibodies were more likely to develop allograft rejection that might be predicted early following transplantation. Algorithms, on the basis of our approach, could be tested for influence of post-transplant allosensitization on graft survival.Su2.116. Humoral and Cellular Response to Influenza Vaccination in Human Recipients Naturally Tolerant to a Kidney Allograft.G. Roussey-Kesler, 1 C. Ballet, 1 J. T. Aubin, 2 S. Brouard, 1 J. P. Soulillou. 1 Background: A rare cohort of kidney recipients continue to enjoy a normal renal function years after interruption of their immunosuppressive treatment and are considered as btolerantQ. To assess wether this state of tolerance is specific to their graft and not the result of a state of immunodeficiency, we studied the immune response of these patients following influenza vaccination. We compared this response to that of kidney recipients under conventional maintenance immunosuppression and to healthy volunteeers.Patients and Methods: 4 tolerant recipients (TOL), 5 immunosuppressed-recipients (IS) and 9 healthy volunteers (HV) received a trivalent influenza vaccine (A/Moscow/10/99; A/NewCaledonia/ 20/99; B/HongKong/330/01) during the period of 2003-2004. The 3 groups were matched for age and renal function (mean age: 49 F 22, 48 F 14 and 48 F 14 years for TOL, IS and HV respectively; mean creatininemia: 104 F 7.2Amol/l for TOL, 108 F 16,8Amol/l for IS). All IS recipients received a conventional immunosuppressive treatment, associating a calcineurin inhibitor with mycophenolate mofetil. The humoral response was measured by hemagglutination inhibition (HI) titers before vaccination and after 1 and 3 months. A positive response was defined as a 4 fold increase in HI titers. During the period of 2004-2005, 4 TOL, 8 IS and 9 HV received a trivalent influenza vaccine (A/Fujian/411/2002, A/Newcaledonia/20/99, B/ Shanghai/361/2002). The cellular immune response was analyzed before and 1 month after vaccination. The frequency of specific T cells was determined by IFNg-secreting T cells detected with an Enzyme-Linked-Immunosorbent Spot (ELISPOT) assay after a 24 hour in vitro stimulation with the vaccine.Results: According to the viral strain, a positive humoral response was observed in 25 to 75% of TOL, in 0 to 40% of IS and in 33 to 89% of HV. Thus, IS recipients presented a poor humoral response as compared to HV, reaching a significant difference for the A/NewCaledonia strain (P b 0.05), whereas the humoral response for TOL was not statistically different from HV. However, 1 month after vaccination, 87% of IS presented a strong cellular response to the influenza vaccination, whereas a comparable positive response was observed only in 50% of TOL and 55% of HV (not statistically different). Taken together, these data suggest that the patients who are tolerant to their kidney respond to the vaccination. In addition we show that the frequency of cells producing IFNg following vaccination is unusually high, possibly due to repetitive stimulations.Conclusion: TOL recipients present a humoral and cellular response to influenza vaccination similar to HV, suggesting that the tolerance state of this small cohort of patients is not related to a global immunodeficiency. Pharmacodynamic Monitoring of Calcineurin Inhibitors by Quantitative Analysis of NFAT-Regulated Gene Expression.T. Giese, 1 M. Schoels, 1 T. Dengler, 2 M. Zeier, 3 S. Meuer. 1 1 Immunology, University of Heidelberg, Heidelberg, Germany; 2 Cardiology, University of Heidelberg, Heidelberg, Germany; 3 Nephrology, University of Heidelberg, Heidelberg, Germany.With the introduction of calcineurin inhibitors (CNI) long-term allograft function has significantly improved. The problem of limited therapeutic margins and the toxicity of CNI remain unsolved. The quantitative assessment of inhibition of NFATregulated gene expression 2 hr after Cyclosporine A (CsA) intake represents a novel approach to evaluate the biological effectiveness of CsA therapy and provides means to enable individualized immunosuppressive regimens.In 55 patients carrying heart allografts we compared the degree of inhibition of IL-2, IFN-g and GM-CSF gene expression with the peak blood concentration of CsA. Functional immunosuppression as assessed by RT-PCR varies considerably among CsA treated individuals with stable graft function. Given the relatively constant level of inhibition over a broad range of drug concentrations, we felt that a considerable group of patients with unnecessarily high CsA doses might benefit from a reduced dosing of the drug without compromising the efficacy of the immunosuppressive therapy. Therefore, we started a clinical study reducing the dosage of CsA with close pharmacodynamic monitoring of the patients. Six patients after kidney transplantation enrolled in this study were monitored over the period of more than one year so far. In all patients the doses of CsA could be safely reduced without significantly changing the level of immunosuppression.In conclusion, patients treated with calcineurin inhibitors might benefit from a reduced dosage of the drug, if they respond to CNI with a strong inhibition of NFAT-regulated gene expression. Profiling of bOperationally TolerantQ Kidney Recipients Using SELDI-TOF Mass Spectroscopy. Christophe Braud, 1 Alexandre DuPont, 1 Magali Giral, 1 Jean-Paul Soulillou, 1 Sophie Brouard. 1 1 INSERM U643, ITERT-CHU Hotel Dieu Nantes, Nantes, France.Despite the discovery of potent immunosuppressive agents, chronic rejection remains the main cause of graft loss after solid organ transplantation. In addition, exposure to immunosuppression may cause infections and malignancies which contribute to the high level of post-transplant morbidity. Achieving clinical tolerance would represent a major progress in transplantation. Operationally tolerant patients, accepting their graft in an imunosuppressive free environnement after clinical organ transplantation, are still extremely rare but represent a unique opportunity of identifying tolerance fingerprints. Surface Enhanced Laser Desorption Ionization Time-of-Flight (SELDI-TOF) mass spectroscopy analysis was used here to profile sera from drug-free tolerant kidney recipients (n = 7) compared to recipients undergoing chronic rejection (n = 8) and control nongrafted patients with renal failure related to bnon-immunologicQ kidney diseases (uropathy, diabetis, n = 8) and whose renal function match that of recipients with chronic rejection Sera were fractionated into 6 fractions and each fraction was loaded on 2 different chromatographic surfaces (metal affinity IMAC30-Cu 2+ and cation exchange CM10). Results were cross validated by analysing serum samples from each individual in two independent experiments. Four protein peaks of interest were selected in 3 out of 6 fractions on the 2 chemistries. The 3 first protein peaks were found significantly increased in sera from patients with chronic rejection and renal failure controls compared to operationally tolerant patients (P b 0.05) suggesting that these protein peaks may be related to renal failure. However, interestingly, the fourth protein peak was increased specifically in sera from operationally tolerant patients (P b 0.05) but neither found in sera from patients with chronic rejections nor in renal failure controls. Considering the absence of specific tolerance markers, the identification of a non invasive and specific biological signature of tolerance would open new perspectives for managing immunosuppressive drugs in long term recipients. Mega Dose Allogeneic Hematopoietic Stem Cell Transplantation, Natural Suppressor Cell Chimerism and Tolerance in Clinic-Ahmedabad Experience.be found in only 1/4 instances. In stable patients, the ATP deviation from the preoperative baseline, indicative of quiescence, was much smaller than that of CNI levels (means of deviations 0.4 F 36 % vs. 18 F 56 %).Conclusion: Immuknow is better correlated with the clinical status than CNI level and therefore could be recommended for post-transplant monitoring. OBJECTIVE: Acute vascular rejection (AVR) and cellmediated rejection (CMR) remain the primary immunological barriers to successful xenotransplantation. While the CD86, but not CD80, costimulatory pathway has been shown to play a critical role in allotransplantation heart allograft rejection, the immunoregulatory roles of CD80 and CD86 have not been fully dissected in xenotransplantation.METHODS: Using a concordant Lewis rat-to-mouse heterotopic heart xenotransplantation model we characterized the role of CD80 and CD86 in xenotransplantation using CD80 and CD86 knockout mice on the C57BL/6 background (Jackson Labs). Xenoantibody levels were measured by flow cytometry by incubating sera from transplant recipients with Lewis rat lymph node cells, followed by staining with anti-mouse IgM, IgG1 or IgG2a -FITC or -PE conjugated antibodies. C3dg intragraft deposition was characterized by western blotting with a goat-anti-mouse C3 polyclonal antibody.RESULTS: C57BL/6 recipients reject xenograft hearts on POD17-21 and show CMR/AVR histopathology. We now report that CD80 À/À recipients reject xenograft hearts on POD5 and show AVR histopathology (an antibody driven process). In contrast, CD86 À/À recipients reject xenograft hearts on POD17 but show predominantly CMR pathology (T cell driven process). We show that CD80 À/À recipients have significantly increased serum levels of IgG1/2a xenoantibodies, as well as intragraft IgG deposition, than CD86 À/À or C57BL/6 recipients at POD5-6. Furthermore, C57BL/6 but not CD86 À/À recipients show an increase in IgG1/2a xenoantibodies on POD17-21. Furthermore, CD80 À/À recipients showed deposition of complement C3dg protein in the graft on POD5, whereas CD86 À/À and C57BL/6 recipients on POD6 or POD17-21 did not show any C3dg deposition.CONCLUSIONS: This data demonstrates that CD80 suppresses xenogeneic antibody-driven AVR, while CD86 promotes CMR. Furthermore, this data suggests that CD80 costimulatory molecule suppresses xenogeneic humoral responses by inhibiting the generation of C3dg, a result of C3 activation. We contend that CD80 and CD86 play distinct roles in regulation of xenogeneic rejection by differentially regulating B cell responses and complement activation/degradation. These results highlight the importance of different mechanisms regulating xenogeneic and allogeneic graft rejection. Our goal is to establish dendritic cell (DC)-based protocols for vaccination and adoptive immunotherapy for refractory PTLD encountered in IS SOTx patients. DC1) generated from IS SOTx patients and healthy donors to boost Type-1 (IFN-g) EBV-specific CD8+ T cells ex vivo. Methods: EBV+/HLA-A02+ IS SOTx patients receiving chronic 2 drugs maintenance therapy (n = 9) or healthy controls (n = 10) were recruited for this study. The EBV-specific CTLs were generated ex vivo by co-culturing autologous T cells with DC1 loaded with a mixture of three peptides derived from EBV Ags. To assess the incidence and the functional polarization of CD8+ T cell responses to EBV epitopes, ELISPOT assays for IFN-g and IL-5, and ELISA assays for TGF-b and IL-10 were performed on PBMC, or on ex vivo generated EBV-specific CTLs. Results:PatientsT peripheral blood circulating CD8+ T lymphocytes preserved their functional Type-1 polarization against EBV Ags, as compared to healthy donors. After 10 days of ex vivo stimulation with EBV peptide-pulsed DC1, the patientsT cocultures contained lower numbers of CTLs, as compared to healthy controls, suggesting a defect in patientsT T cell proliferative potential. However, IFN-g producing EBV-specific CD8+ T cells were successfully boosted in both IS SOTx patients and normal controls, at comparable frequencies, and this appeared to reflect an expansion of pre-existing EBV bmemoryQ T cells. In addition, the patientsCTLs produced significantly higher levels of IL-10 (but no TGFb) as compared to healthy donors, suggesting that some regulatory T cells may have been expanded in vitro. Conclusion: Our results show that IS stable SOTx patients display Type-1 immune (IFN-g) responses against EBV Ags in their peripheral blood, to a degree comparable to that observed in normal controls. These T cells can be further boosted ex vivo using EBV Ag-loaded Type-1 polarized DCs. However, only the patientsT expanded co-cultures contained EBV-specific CD8+ T cells producing IL-10. These data suggest that patientsT (but not normal donor) DCs might be partially impaired in their ability to become fully matured, since they elicited both IFN-g and IL-10 production from EBV-specific CD8+ T cells. We are currently investigating approaches to expand Type-1 biased T cells in DC-based co-culture conditions, for the optimization of adoptive immunotherapy of PTLD in SOTx patients.

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II Paper ID: d89a1a60a3873bbcf68b3ec0ffcb1fc3cb276205 (Document no.: 24094)

III Authors: Authors not available

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Poster Prize Award Ceremony and Closing Chairs: T. Yamamoto, Japan/K. Arimura, Japan Plenary Lectures PD-1 belongs to the CD28/CTLA-4 co-receptor family and negatively regulates immune responses upon interaction with its ligands, PD-L1 and PD-L2. PD-1 −/− mice develop various autoimmune diseases depending on their genetic background. On BALB/c background, PD-1 −/− mice develop autoimmune dilated cardiomyopathy (DCM) with high-titer autoantibodies against 30 kDa cardiac antigen. We have recently identified the 30 kDa antigen as cardiac troponin I (cTnI) and showed that the deregulated production of anti-cTnI autoantibody is the main cause of DCM in PD-1 −/− mice.More recently we analyzed the function of FcγRIIB in the DCM of BALB/c-PD-1 −/− mice, because FcγRIIB has been known to play pivotal roles in the removal and inhibition of autoreactive B cells. DCM of BALB/c-PD-1 −/− mice was not affected by the disruption of FcγRIIB, however BALB/c-FcγRIIB −/− PD-1 −/− mice developed autoimmune hydronephrosis with concomitant production of autoantibodies against urothelium. Massive inflammation was observed around the pelvis of the kidney and at the junction between the ureter and the bladder, which were considered to be the cause of the occlusion and the subsequent development of hydronephrosis. BALB/c-FcγRIIB −/− PD-1 +/− mice developed hydronephrosis with the similar frequency as BALB/c-FcγRIIB −/− PD-1 −/− mice, whereas BALB/c-FcγRIIB −/− mice did not show any autoimmune symptoms. Therefore the autoimmunity to urothelial cells seems to be controlled primarily by FcγRIIB and exaggerated by PD-1 deficiency.These observations suggest the collaborative role of FcγRIIB and PD-1 in the regulation of autoimmunity against urinary tract but not heart and may help understand the multi-genic nature of the autoimmune diseases. Shizuo Akira Research Institute for Microbial Diseases, Osaka University, and Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency, Suita 565-0871, JapanMammalian Toll-like receptors play a critical role in detection of invading pathogens as well as triggering of subsequent inflammatory and immune responses. Individual TLRs recognize different microbial components. Adaptor MyD88 is essential for the responses to all TLR ligands except for TLR3 ligand, and is involved in induction of inflammatory cytokines through activation of IRAKs and TRAF6. In addition, the TLR3-and TLR4-mediated signaling possesses a MyD88independent pathway, which is activated by another adapter molecule, TRIF. The TRIF-dependent pathway activates IRF3 through TBK1, which results in induction of type 1 interferon. Plasmacytoid dendritic cells (pDC) rapidly produce IFN alpha in response to viral infection or in response to TLR7-(imidazoquinolines, sRNA) or TLR9-ligand (CpG DNA). Such IFN alpha induction by pDC involves formation of a complex consisting of MyD88, TRAF6, IRAK-1 and IRF7, in which IRAK-1 acts as IRF7 kinase. Besides TLRs, recent findings have shown the presence of cytosolic detector system of invading pathogens. Two DExD/H box RNA helicases, retinoic acid inducible protein-I (RIG-I) and melanoma differentiation-associated gene 5 (mda-5) are involved in antiviral responses by recognizing dRNA in the cytoplasm. RIG-I and mda-5 recognize distinct RNA viruses. Recently, we have generated IPS-1 knockout mice, and shown the essential role in RIG-I and mda-5 dependent signaling pathways. In this symposium I will discuss recent progress in the TLR-dependent and independent recognition of viral infections.Compared with autoimmune T cells or B cells, much less attention has been paid onto the role of NK cells in multiple sclerosis (MS) . Although speculative, this could be explained by limited understanding of NK cell biology some time ago, or possibly due to some controversies in the literature. However, as NK cells are a major source of regulatory cytokines such as IFN-γ and because they would functionally be polarized towards NK1 or NK2, their involvement in the pathogenesis of autoimmune diseases like MS is thought to be most likely. In fact, recent works from our laboratory have showed that NK cells in the peripheral blood of MS are remarkably altered regarding cytokine secretion profile and surface phenotypes (Takahashi et al. Our initial key observation was that NK cells in remission of MS would exhibit the properties of NK2 cells, with some similarities to Th2 cells. This change is likely to be protective against MS and can be interpreted as an adaptive change to maintain the state of remission. Further analysis has supported this idea and also convinced us that "Peripheral NK cell is a window into the disease status of MS." For example, we have recently found that a higher expression of CD11c on NK cells predicts an occurrence of relapse within a few months, whereas absence of CD11c and CD95 on NK cells would define the most stable remission (Aranami et al. In terms of the mechanism of how NK cells would mirror the disease status, I will present our hypothetical model and discuss on the implications and the future direction of research. DR01-03 Heat shock protein-60 and autoimmunity Irun R. Cohen Department of Immunology, Weizmann Institute of Science, Rehovot, IsraelHeat shock proteins (HSP) were initially identified as a family of stress-induced proteins characterized by their chaperone activity. However, they were later identified as self-antigens recognized by host T cell and autoantibodies in many autoimmune diseases. Recently, the role of HSP as endogenous activators of the innate immune system has been unveiled. Here, I shall discuss the relevance of HSP60 for the regulation of inflammation and autoimmune disease. Its pro-inflammatory activity derives from its innate TLR4-dependent activation of macrophages and dendritic cells; microgram/ml concentrations are required. Its anti-inflammatory activity results from 3 signaling functions: (1) Epitopes of HSP60, complexed to MHC-II, are expressed by activated effector T cells; these complexes activate anti-ergotypic regulatory T cells. (2) HSP60 is an innate activator (via TLR4) of antigen-presenting B cells which then secrete IL10 and polarize responding T cells towards Th2. (3) HSP60, nanograms/ml suffice, acts as a co-stimulator of CD4 + CD25 + Tregs and enhances the ability of relatively low concentrations of the Tregs to down-regulate CD4 + CD25 − or CD8 + target T cells, detected as inhibition of target T-cell proliferation and IFN-γ and TNF-α secretion. The anti-inflammatory effects of HSP60 on T and B cells can account for its success in down-regulation of autoimmune diseases such as human or mouse Type 1 diabetes mellitus and rat adjuvant arthritis. A peptide of HSP60 is currently in phase 3 trials in new-onset human diabetes. Migration studies suggest that some event(s) early in life primes genetically susceptible individuals for MS later in life. Also different viral infections are associated with exacerbations of MS. However, no single virus has been identified as the causative agent of MS. We have developed a model where infection of mice with a virus having molecular mimicry can prime these mice for later disease induced by a different virus. After infection when recombinant VV PLP has been cleared, mice have no clinical signs of experimental autoimmune encephalomyelitis (EAE). When VV PLP primed mice are then challenged with murine cytomegalovirus, mice develop exacerbations of EAE. These data indicate that viruses having molecular mimicry could prime genetically susceptible animals for disease early in life that can be induced by a different virus later in life. DR02-01 Mechanisms of immune-mediated demyelination in the CNS J.P. Antel, MD McGill University, Montreal, Canada The capacity of immune mediated mechanisms to mediate demyelination within the CNS has been recognized since the description of cases of acute disseminated encephalomyelitis (ADEM) that followed the introduction of the neural tissue containing Pasteur rabies vaccine in the 1880s and the subsequent development of its animal model experimental auto-immune encephalomyelitis (EAE). These disorders indicate the requirement for auto-reactive CD4 T cells to initiate an inflammatory response within the CNS. The immune cells and molecules that actually mediate injury of myelin or its cell of origin the oligodendrocyte (OLG) likely extend beyond these inflammation initiating cells and include constituents of the adaptive and innate immune systems. Selective neural target injury could reflect the properties of the immune effectors and/or the target cells. Selective immune effectors would include OLG/myelin specific MHC class 1 restricted αβ CD8 T cells and antibody. Injury mediated by members of the tumor necrosis factor (TNF) superfamily (fas, TRAIL, TNF) provide examples of target-conferred selectivity dependent on expression of the requisite receptor. A chronic inflammatory environment can contribute both to enhanced production of such effector molecules and upregulation of their receptors. Such an environment can also enhance expression of molecular partners (CD56, NKG2D and their ligands) involved in non MHC restricted cytotoxic responses mediated by innate immune cells (NK cells, γδ T cells) and even induce such molecules on αβ T cells. Neuro-protective strategies can be directed at the immuneneural molecules directly involved in the injury process or at modulating the CNS environment that regulates their expression and/ or activity. DR02-02 Neuropathology of multiple sclerosis H. Lassmann Center for Brain Research, Medical University of Vienna, Vienna, Austria Multiple sclerosis (MS) has initially been defined as a chronic inflammatory disease of the central nervous system, which gives rise to focal demyelinated plaques in the white matter. Focal white matter plaques are highly heterogeneous regarding the immunopathological profile, the patterns of tissue destruction and their capacity for repair. This heterogeneity is in part stage dependent and in part reflects a true diversity between patients or patient subgroups. In addition, pathological alterations are not restricted to focal white matter plaques, but the CNS is affected in a global sense. In such patients extensive demyelination is also found in the cerebral cortex and in the deep brain stem nuclei. Furthermore, there is massive diffuse axonal injury and damage in the normal appearing white matter, which is associated with persistent inflammation by T-cells, B-cells and Plasma Cells as well as by global and diffuse microglia activation. Only part of the spectrum of pathological alterations within the MS brain can so far be detected by magnetic resonance imaging. In addition, the pathogenesis of the diverse components of MS pathology is apparently different. The complex pathology of MS suggests that therapeutic strategies should be tailored to cope with these stage and patient dependent variability. There is an intense cross-talk between muscle and immune cells. Muscle cells can express many immunologically important molecules, including MHC and co-stimulatory molecules. Furthermore, muscle cells can secrete many cytokines, chemokines and cell adhesion molecules, as well as receptors of the innate immune system. Conversely, elements of the immune system affect muscle cell properties. Therefore, muscle cells should be considered as active modulators rather than passive targets of immune reactions. This is relevant to the immunopathogenesis of the different inflammatory myopathies. In polymyositis and inclusion body myositis, clonally expanded CD8-positive T cells invade muscle fibers that express MHC class I antigens. Recently, however, it has become possible to "revive" putatively pathogenic, autoimmune T cells from frozen specimens of human muscle biopsy tissue. Using CDR3 spectratyping, clonally expanded T cell receptor (TCR) β-chains can be identified in muscle sections of patients with inflammatory myopathy. The putatively pathogenic T cells can be isolated by laser-microdissection, and co-expressed pairs of TCR alpha-and beta-chains identified by a multiplex PCR protocol which allows the concomitant amplification of both chains from single cells. The paired TCR alpha-and beta-chains are functionally expressed in T-hybridoma cells, which can be used to search for the target antigen(s).Refs. Today 15: 269-274, 1994; Wiendl, Hohlfeld, and Kieseier, Trends Immunol. DR02-04 Immune mediated neuropathies Department of Neurology, Heinrich-Heine-University of Duesseldorf, Germany The peripheral nerve is targeted by aberrant immune responses in a number of acute and chronic neuropathies including Guillain-Barre Syndrome, CIDP, paraproteinemic neuropathies, vasculitic and paraneoplastic neuropathies.Local immune circuitry is made up of endoneurial macrophages and Schwann cells. The PNS is separated from the systemic immune compartment and blood by the blood-nerve barrier which is however absent or deficient at its most proximal and distal parts where therefore immune cells, antibodies and mediators can readily access neural structures. Principles of normal immune reactivity in nerve and responses to infections and danger signals will be reviewed. These concepts will be illustrated with reference to Guillain-Barre Syndrome and CIDP. In both predominantly the myelin sheath is attacked by disturbed immunity. Glycolipid antibodies have been implicated in dysfunction and structural damage in GBS and its variants whereas myelin proteins have been identified in a number of patients with CIDP as target autoantigens. The concept of molecular mimicry has been elaborated in depth in GBS which is often antedated by infections with Campylobacter jejuni or cytomegalovirus. There will be a discussion of new transgenic models representing mechanisms determining spontaneous initiation, the course of CNS autoimmunity, the distribution of lesions within the CNS and the cellular composition of the inflammatory infiltrate. Then I shall review aspects of the early, inflammatory phase of MS plaque generation, in particular concerning the dynamics of immune cell invasion into the central nervous system (CNS). PS02-01 Regulatory function of NKT cells in the protection of autoimmune disease development Masaru Taniguchi, Ryusuke Nakagawa, Satoshi Kojo, and Hiroshi Watarai RIKEN Research Center for Allergy and Immunology, Yokohama, Japan NKT cells produce large amounts of both IFN-γ and IL-10 at the same time and thus mediate dual functional activities, including protective and regulatory functions. When NKT cells are in vivo activated with a single injection of α-galactosylceramide (α-GalCer), splenic DCs undergo a rapid maturation and acquire a potential to produce high IL-12, which induces Th1-type response. By contrast, repeated α-GalCer-stimulation activates NKT cells to keep producing IL-10 but stop producing IFN-γ, resulting in generation of regulatory DCs, which produce high IL-10 and low IL-12. In fact, IL-10 from NKT cells in the absence of IFN-γ keeps maintaining regulatory activity of DCs to produce IL-10 for a long time (at least 30 days), and also generates IL-10-producing regulatory T cells. The high IL-10 and low IL-12 production in the regulatory DCs are due to the upregulation of phosphorylation of p44/42 extracellular signal-regulated kinase (Erk-1/2) and the augmented production of IκB induced suppression of NFκB activation, respectively. When experimental allergic encephalomyelitis (EAE) is induced, EAE development is significantly suppressed by generating regulatory CD4 + T cells which are induced by the transfer of myelin oligodendrocyte glycoprotein-pulsed DCs obtained by repeated α-GalCer-stimulation. These findings indicate that NKT cells play important roles in inducing regulatory T cells suppressing EAE development. Invariant NKT cells in autoimmunity Sachiko Miyake Department of Immunology, National Institute of Neuroscience, NCNP, Japan CD1d-restricted invariant Vα14 (Vα14i) NKT cells are a unique lymphocyte subset implicated in a variety of immunoregulation. Vα14i NKT cells inhibit disease in some autoimmune disease models such as experimental autoimmune encephalomyelitis (EAE) and type I diabetes whereas they exacerbate disease in other models such as arthritis and allergic airway inflammation. Even though the role of Vα14i NKT cells in the pathogenesis is reciprocal in these models, activation of Vα14i NKT cells with synthetic glycolipid ligands suppresses these diseases. OCH, sphingosine truncated derivatives of α-galactosylceramide (α-GalCer), stimulate Vα14i NKT cells to selectively produce Th2 cytokines and prevent Th1-mediated autoimmune disease models. The other derivatives of α-GC suppressed antibody-induced arthritis and allergic airway inflammation. These results highlight the therapeutic potential to target Vα14i NKT cells in the treatment of these diseases.Recently an invariant Vα19-Jα33 T cell receptor α-chain (Vα19i) expressing, MR1-dependent cell population emerged as an second invariant NKT cell population. Vα19i NKT cells, however, have an unidentified role in autoimmunity. We demonstrate that over-expression or adoptive transfer of Vα19i NKT cells inhibited EAE. Conversely, MR1deficient mice, lacking Vα19i NKT cells, exhibited exacerbated disease.The mechanisms of Vα19i NKT cell-mediated suppression of disease will also be discussed. PS02-03 Mucosal associated invariant T cells: An evolutionarily conserved T cell subset restricted by MR1 O. Lantz a, b , L. Duban a, b , I. Cruz-Moura a, b and E. Treiner c a Laboratoire d'Immunologie, Institut Curie, Paris, France; b Inserm U653, Paris, France; c Inserm E0351, Amiens, FranceThere are only two T cell subsets displaying a conserved invariant repertoire among species, NK-T cells and the recently discovered Mucosal Associated Invariant T (MAIT) cells both of which express highly conserved TCRα chains (iVα24/14-Jα18 and iVα7.2/19-Jα33, respectively) and recognize related ligands (CD1d and MR1, respectively) in human and mouse.MAIT cells are -Highly abundant in cattle blood, human blood and gut and less so in mouse gut, -Selected by B cells and not by the thymic epithelium, -Restricted by MR1, a strikingly phylogenetically conserved MHC class Ib molecule. -TAP and Ii chain independent.-Dependent upon the commensal microbial flora (they are absent from germ free mice). -Secrete high levels of TH2 cytokines (IL-4, IL-5 and IL-13).The function of MAIT cells is unknown but the high conservation of both the TCRα chain and the MHC ligand between species suggests an important one, such as gut epithelial homeostasis or control of the class of the immune response in the gut. Alternatively, MAIT cells could be involved in the defense against ubiquitous pathogens.Importantly, accumulation of MAIT cells have been found in the lesions of multiple sclerosis (MS) patients as well as in nerve biopsy samples from chronic inflammatory demyelinating polyneuropathy (CIDP) patient. PS02-04 The response to self lipids in autoimmune diseases G. De Libero Experimental Immunology, Department of Research, University Hospital, Basel, Switzerland Recognition of self is essential for repertoire selection, immune regulation and autoimmunity. It may be a consequence of infection, representing the escape strategy adopted by pathogens but also inciting autoimmunity.Immune response to self lipid antigens is a novel mechanism that may contribute to autoimmune diseases. T cell recognition of self lipids is influenced by the repertoire of immunogenic self lipids, the rules of lipid presentation by CD1 isoforms and by the regulation of lipid metabolism. The modifications occurring in lipid structure during ontogeny, synthesis in different organs and in inflammation may lead to generation of highly immunogenic self lipids and to the efficient activation of autoreactive T cells.Bacterial infection may also have dramatic effects on lipid metabolism and on lipid antigen presentation. This is illustrated by enhanced de novo synthesis of immunogenic self lipids in infected cells which thus become stimulatory for self lipid-reactive T cells.The mechanisms regulating T cell response to self lipids will be discussed in the context of the autoimmune response in Multiple Sclerosis. PS03-00 The Admixture Multiple Sclerosis Susceptibility Locus on Chromosome 1 is refined to a 4 Mb Interval in an African-American PopulationThe HLA DRB1*1501 haplotype is the only well-validated genetic risk for MS. However, we recently published a genome-wide admixture scan that identified a second locus associated susceptibility for MS in 582 African-American patients (LOD score 5.2). Control subjects showed no significant difference in the level of European ancestry at this locus. We are expanding our collection of African-American subjects with MS and have now studied 654 subjects using our admixture mapping strategy. We are also performing fine-mapping analyses by comparing our affected subjects with over 500 African-American control subjects. The addition of subjects and markers to our published dataset has improved the maximal association to a LOD score of 6.9 and has refined our locus from an 8 Mb to a 6 Mb segment. Efforts are ongoing to further refine this locus; an additional 200 affected subjects and 500 markers are currently being genotyped. We are rapidly refining the location of this second MS susceptibility allele; 50 genes lie in the current interval of 6 Mb. Finding this risk allele will be particularly informative for African-American patients with MS as it may identify a new molecule or pathway involved in the pathophysiology of MS. This work may therefore generate new targets for therapy and may be broadly applicable to all patients with MS. PS03-01 Whole genome association screen in multiple sclerosis DAS Compston on behalf of The International Multiple Sclerosis Genetics Consortium (IMSGC)⁎ Early candidate gene studies quickly identified the importance of the extended human leukocyte antigen (HLA) DR15 haplotype in conferring susceptibility to multiple sclerosis. But subsequent whole genome linkage screens have failed conclusively to identify any other susceptibility factors. Results from the most recent high-density linkage screen indicate that non-HLA loci exert only modest effects individually, and will require testing for association in large datasets if they are to be identified with any statistical confidence. One often considered consequence of LD is the possibility effectively to screen very nearly all the common variations in the human genome by typing just a proportion of these polymorphisms, especially if the typed markers are carefully chosen to capture as much of the available variation as possible (e.g. The technology required to undertake such "indirect" whole genome association screening is now a reality. In our indirect association screen for multiple sclerosis the International Multiple Sclerosis Genetics Consortium (IMSGC) is genotyping the Affymetrix 500k chip in 1000 trio families (an affected International Symposium: Alzheimers disease, neurodegeneration and immunity Alzheimer's disease (AD) is an age-related progressive neurodegenerative disorder characterized by memory loss and severe cognitive decline. These clinical features are manifested morphologically by excessive accumulation of extracellular amyloid β-peptide (A-beta) in the brain parenchyma, particularly in the hippocampus and cerebral cortex, leading to neuronal loss. In addition, aggregates of A-beta that forms neuritic plaques in the brain become toxic in that they trigger chronic glial activation and thereby interfere with normal brain function. Investigating how the immune system is involved in the disease is based on new emerging characteristics of three biological systems: autoimmunity, brain-immune interactions, and neurogenesis. Our data demonstrate that A-beta specific T cells are induced in AD and that the specificity and magnitude of these responses depend primarily on HLA-DR alleles. Limited expression of IFN-γ in the brain, as observed during normal brain aging, is essential to promote migration of these A-beta reactive T cells to the parenchymal tissue in the hippocampus and subsequently interaction with local antigen presenting cells. In contrast to the injurious effects induced by chronic innate immune mechanisms involved in AD, T-cell-mediated immunomodulation may support tissue recovery by regulating glial activation and differentiation, clearance of A-beta, and neuronal survival. The translation of these results to safe immunotherapeutic approaches will be discussed. IS01-04 Mucosal immunotherapy for Alzheimer's disease with viral vectors H. Hara a , M. Inoue b , M. Hasegawa b , Y. Yonemitsu c , T. Nabeshima d and T. Tabira a a National Center for Geriatrics and Gerontology, Obu, Japan; b DNAVEC corporation, Tsukuba, Japan; c Kyushu University, Fukuoka, Japan; d Nagoya University, Nagoya, Japan Background: Alzheimer's disease is characterized by progressive loss of cognitive function due to amyloid-β (Aβ) deposits in the central nervous system. Based on the amyloid cascade theory, many reports indicated that immunotherapy is effective for the treatment of Alzheimer's disease. We developed the mucosal immunotherapy for Alzheimer's disease by nasal administration of recombinant sendaivirus vector expressing Aβ 1-43/IL-10. Method: 24-25-months old APP transgenic mouse were nasally administered once with recombinant sendaivirus vector expressing Aβ 1-43/IL-10. After 8 weeks of treatment, mice were sacrificed and investigated the amyloid burdens in the brains by immunohistochemistry and ELISA. Results: Serum antibody levels for Aβ were elevated after four weeks of treatment. Immunohistochemistry of the mouse brain tissue showed that the extra-cellular amyloid deposits were clearly decreased compared to the nontreated mouse. The inflammation and lymphocytes infiltration were not recognized in each organ including brain. Conclusion: Nasal administration of recombinant sendaivirus vector expressing Aβ 1-43/IL-10 is advantageous in that no cellular immune response was elicited and only antibody production was induced, furthermore, no adjuvant is required. This new mucosal immunotherapy does not induce the strong immune reactions and can reduce the side effects of encephalitis as found in Aβ peptides vaccination. IS01-05 Antibody independent clearance of A-beta in a mouse model of Alzheimer's disease by activation of microglia via nasal vaccination Methods: Age-and sex-matched littermates (APP-J20 mice) received a weekly nasal treatment with Protollin (a proteosome-based adjuvant used in human influenza vaccination) 1 μg/treatment, plus glatiramer acetate (GA, an approved drug for MS) 25 μg/treatment, beginning at age 5 months and were treated for 8 months. Results: We found a significant reduction in amyloid fibril (∼ 80%) in GA + Protollin treated mice versus controls (P < 0.001). There was reduction of both (50%) soluble Aβ and (49%) insoluble form of Aβ (p < 0.02) as measured by ELISA and elevation of total serum Aβ levels. Clearance of Aβ was also observed with nasal Protollin alone and direct intrahippocampal injection of Protollin activated microglia and reduced Aβ plaques. We previously reported that nasal vaccination with GA + Protollin, decreased Aβ plaques in 14 month old APP Tg mice, vaccinated animals developed activated microglia (CD11b + cells) that co-localized with Aβ fibrils, and the extent of microglial activation correlated with the decrease in Aβ fibrils (JCI: 115:2423 (JCI: 115: , 2005 . In this study, after chronic treatment for 8 months we observed no difference in microglial activation, suggesting that once Aβ is cleared there is downregulation of microglial activation. Conclusion: Our results demonstrate an antibody-independent therapeutic approach for the treatment of AD which targets microglial cells and is also effective, without toxicity, when given chronically to young mice as prevention. Beers, Q. Xiao, and W. ZhaoMethodist Neurological Institute, Houston, USA Immune/inflammatory factors have been implicated in the pathogenesis of motoneuron degeneration in amyotrophic lateral sclerosis with the presence in spinal cord tissue of activated microglia, dendritic cells, IgG deposits, lymphocytic infiltrates, and marked increases in MCP-1, the chemokine involved in immune cell recruitment. Transgenic mice overexpressing mutant human superoxide dismutase (mSOD1) provide a useful model to investigate mechanisms of motoneuron injury. In such mice, activated microglia and increased MCP-1 are present prior to onset of weakness; and dendritic cells subsequently appear. To define the potential role of microglia in disease pathogenesis, we used a mouse model unable to develop myeloid cells or lymphoid cells (PU.1 −/− ). Bone marrow transplants of these mice result in donor-derived microglia in spinal cord parenchyma. Transplantation of mSOD1 bone marrow alone did not induce disease. However, PU.1 −/− mice with mSOD1 expressed in motor neurons and transplanted with wild type mSOD1 donor cells had greater motoneuron preservation and prolonged disease duration and survival compared with mice receiving mSOD1 donor cells. In vitro studies confirmed that wildtype microglia were less neurotoxic than mSOD1 microglia. Transplant of these mSOD1/PU.1 −/− mice with bone marrow lacking the receptor for MCP-1 (CCR2 −/− ) removed the neuroprotective effects of wildtype microglia. When mSOD1 was expressed in RAG2 −/− mice, which lack functional T-and B-cells, disease progression was actually accelerated. These studies suggest that CNS parenchymal microglia can be neuroprotective or toxic, with the specific effects modulated by communication with peripheral immune cells. IS02-02 Neuroinflammation and non-cell autonomous death of motor neurons in ALS The primary characteristic of Amyotrophic Lateral Sclerosis (ALS) is the premature death of motor neurons. An inherited form is caused by mutation in superoxide dismutase (SOD1). Toxicity is non-cell autonomous, requiring mutant SOD1 acting within motor neurons, neighboring astrocytes and microglia. Reducing mutant SOD1 synthesis in microglia by selective gene excision has little effect on disease onset, but strikingly extends survival after onset, demonstrating that damaged microglia play an essential role in disease progression. Further, using primary motor neuron and microglial cells, only conditioned medium from mutant SOD1 microglial cells produces neurotoxicity on mutant expressing motor neurons. Exploiting these findings, a gene therapy has been developed for targeting almost any gene using continuous infusion to deliver antisense oligonucleotides to neurons and non-neuronal neighbors throughout the nervous system. Oligonucleotide infusion to promote catalytic degradation of the mRNA encoding mutant SOD1 slows disease progression. Thus, antisense oligonucleotide therapy can be an effective means of treating neurodegenerative diseases, including ALS, Alzheimer's, Parkinson's, and Huntington's, for which appropriate target genes have been identified. IS02-03 Phenotypic diversity of microglia and macrophages Institute of Neuropathology, University of Göttingen, Germany Several findings reveal that treatment with certain cytokines, confrontations with microbial agents or exposure to tumor material and apoptotic cells trigger macrophages to acquire distinct reactive phenotypes. Phenotypes are thereby not defined by a single release product or receptor expression, but comprise timed, coordinated, often even reciprocal induction patterns of mediators for cellular communication and profiles of effector molecules (e.g. cytokines, metabolic or extracellular matrix-modelling enzymes, surface structures for phagocytosis or antigen presentation). Adaptations of the executive behavior come with functional consequences, including opposite effects on inflammation, skewing for types of immune responses and orientation for either defense or repair. Although microglia, the CNS tissue macrophage equivalent, is challenged by virtually any neuropathological scenario, less is known concerning the phenotypic diversity and dynamics, associated molecular characteristics and functional consequences. Conceivably, these cells also choose rather variable response options for an adequate coping with an exogenous or endogenous threat and to decide on a recruitment of adaptive immune support. Yet microglia and macrophages of non-CNS origin may differ in reactive profiles as imposed by the specific biochemical milieu, structural vulnerability and control of defence mechanisms in neural tissues. Most notably, distinct reactive phenotypes may not only occur as alternatives, but develop (interchange) stage-specifically during an activation process. Highly organized, context-determined, situation-adapted switches in anti/inflammatory messenger production, timed recruitment vs. decline of immune infiltrates, phagocytotic clearance and tissue rebuilding attempts could be elements of an open, (usually) selflimiting and (mostly) efficient program for protection and restoration. IS02-04 Neurotoxicity by microglia: Mechanisms and potential therapeutic strategy Akio Suzumura Department of Neuroimmunology, RIEM, Nagoya University, Nagoya, Japan Accumulation of activated microglia are observed in or around degenerating neurons. These cells may play a critical role on both neurotoxicity and neuroprotection. Glutamate and TNF-α from activated microglia differentially contribute to neuronal damage in neurodegenerative diseases. However, the clinical trials to block these factors failed in serious adverse effects. Here, we show that TNF-α stimulates extensive microglial glutamate release to cause excito-neurotoxicity, by activating glutaminase. In addition, glutamate is released from microglia through the hemichannels of gap junction, but not through glutamate transporters. Either a hemichannel blocker or glutaminase inhibitors effectively suppressed microglial glutamate release and subsequent neuronal cell death. The unique mechanisms of microglial glutamate release will give us a new strategy against neurodegenerative diseases without perturbation of physiological signals of glutamate. IS02-05 Role of immunity in neurodegeneration in autoimmune encephalomyelitis Institute of Neurology, University College London, London, United Kingdom For many years multiple sclerosis (MS) has been thought to result from autoimmune attack of the central nervous system (CNS). As such, experimental autoimmune encephalomyelitis (EAE)an autoimmune model of MS has greatly influenced the development of immunotherapeutic paradigms aimed at treating MS. Despite some limited success in inhibiting relapsing disease, there has been poor translation of animal studies into the clinic and progressive MS has largely been unresponsive to immunotherapy. However, it has become increasingly recognized that neurodegenerative elements are responsible for disease progression in MS. Axonal loss increases with increasing numbers of neurological attacks and results in the development of permanent residual paresis. As active clinical disease in MS and EAE are associated with mononuclear cell infiltration of the CNS infiltration, demyelination and axonal loss, inhibition of inflammation is one route to neuroprotection and if relapsing disease is inhibited sufficiently early in the disease course, it will slow the rate of atrophy of the CNS and axonal loss. However, when immunotherapy is initiated late into chronic-relapsing disease course, whilst relapsing disease can still be eliminated such as using antigen-specific tolerance induction, there appears to be a slow deterioration in mobility, an increase in neurological signs and ongoing axonal damage that is (auto)immuneindependent. Therefore just as appears to be the case in MS once sufficient damage has been accumulated then inhibition of (auto) immunity via neuroimmunological approaches may be insufficient to control progressive disease elements. IS02-06 Protective effect of mucosal tolerance to E-selectin against memory impairment and white matter injury in a rat model of vascular dementia a Hideaki Wakita and b John M. Hallenbeck a Department of Vascular Dementia Research, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Obu City, Japan; b Stroke Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USAThe subcortical ischemic form of vascular dementia is a common type of vascular dementia, and one of the major causes of cognitive decline in elderly people. Ischemic white matter lesions are the characteristic pathological changes in subcortical ischemic vascular dementia. Ischemic white matter lesions and impairment of memory function have been reported in the rat under chronic cerebral hypoperfusion. Using this model, we investigated the effects of mucosal tolerance to E-selectin on white matter damage and memory impairment. Wistar rats were subjected to the permanent occlusion of both common carotid arteries. Mucosal tolerance was induced by repetitive intranasal administration of E-selectin. Rarefaction of white matter was evaluated by luxol fast blue stain. Memory impairment was evaluated by object recognition, T-maze spontaneous alternation, and T-maze memory retention tests. Mucosal tolerance to E-selectin had a potent protective effect against white matter rarefaction. In addition, mucosal tolerance to E-selectin had a suppressive effect on activation of brain vessels. Mucosal tolerance to E-selectin also protected against memory impairment. These results indicate that the mucosal tolerance to E-selectin suppresses vessel activation support of local immunological and inflammatory processes and protects against ischemic white matter damage. These results can provide a new therapeutic strategy using a mucosal tolerance to E-selectin against subcortical ischemic vascular dementia. IS03-01 Immune regulation of neural stem cell programs in EAE S. J. Khoury Brigham and Women's Hospital, and Harvard Medical School, Boston, MA, USA Multiple sclerosis is characterized by a progressive pathology and alteration in remyelination capacity. Neural stem cells and oligodendrocyte progenitors can generate myelinating oligodendrocytes during development and during normal adult turnover, but endogenous progenitors cells in the MS appear to fail with chronic progression and the responses of endogenous cells in EAE appear to be limited. We hypothesize that inflammatory mediators may act upon the genetic program of NSCs and initiate the process of repair, but in the chronic phase inflammatory mediators may decrease the intrinsic properties of NSCs, resulting in failure of repair. NSCs express immune-related genes that include chemokines and cytokines receptors, and we have shown that some of these molecules such as the chemokine SDF-1 are permissive for the repair function of NSCs during injury.EAE lesions express signaling molecules for neural stem cells, yet there is a lack of neurogenesis and lack of oligodendrogenesis in the chronic phase, and this lack of function is correlated with microglia activation in vivo and in vitro. In order to identify potential mechanisms, we have focused in the molecular biology of candidate genes that may mediate such interactions, we will present data regarding the molecular effects of prototypical Th1 cytokines in the molecular programs of NSCs. Conclusion: Identifying the molecular mechanism for the responses of NSCs to injury in EAE, will help to increase the efficacy of exogenous NSC transplantation and increase the activation of the intrinsic properties of endogenous stem cells in models of multiple sclerosis.Brigham and Women's Hospital, and Harvard Medical School, Boston, MA, USA Dendritic cells are potent antigen presenting cells (APCs) that can mediate tolerance or immunity depending on their state of maturation. Ex-vivo incubation of DCs with CTLA4Ig and MOG peptide alters the biological function of these cells rendering them tolerogenic. Ex-vivo modified DCs have the characteristics of immature DCs, with decreased expression of MHC II, CD80 and CD86, and cytokine production. We show that B7 molecules expressed by DCs are required for the tolerogenic effect, but not Fc-gamma receptors, and the mechanism of protection is independent of indoleamine 2,3dioxygenase. Tolerogenic DCs exhibit decreased expression of surface CD80, CD86, MHC II molecules, and cytokine production. Interestingly, protection from EAE is transferable by APCs but not T cells from the original recipients. Conclusion: Ex-vivo exposure of DCs to tolerizing regimen such as CTLA4Ig may provide a novel therapeutic option with fewer systemic side effects for autoimmune diseases. IS03-02 Neural stem cell transplantation: Cell replacement vs. bystander neuroprotection G. Martino Neuroimmunology Unit, DIBIT -San Raffaele Scientific Institute, Milan, Italy Recent evidence challenges the sole view that neural stem/precursor cells (NPCs) achieve their therapeutic efficacy exclusively by a cellreplacement mechanism. In fact, NPCs may also promote central nervous system (CNS) repair by their intrinsic neuroprotective ability, which are mainly exerted by undifferentiated cells releasing, at the site of tissue damage, a milieu of neuroprotective molecules temporally and spatially orchestrated by environmental needs. This milieu contains molecules (such as immunomodulatory substances, neurotrophic growth factors and stem cell regulators) that are constitutively expressed by NPCs for maintaining tissue homeostasis both during development and in adulthood. The intrinsic nature (pleiotropism and redundancy) of these molecules as well as their 'constitutive' expression by different types of stem cells (such as mesenchymal and haematopoietic stem cells) may represent a stem cell signature. This might also reconcile data showing that other sources of somatic stem cells (such as mesenchymal stem cells and haematopoietic stem cells), with very low potential of neural (trans) differentiation, may efficiently promote CNS repair. Thus, cell plasticity can also be viewed as the capacity of somatic stem cells to adapt their fate and function to specific environmental needs, which arise as a result of pathological conditionsthis is the notion of therapeutic plasticity. The ability of transplanted NPCs to protect the brain from several types of injuries using different and/or multifaceted bystander strategies is of significant importance for the future of stem cell-based therapeutic approaches. IS03-03 T-cell based therapy operates in synergy with adult neural stem cells for neuronal survival and renewal: Implications for Alzheimer's disease Michal Schwartz Department of Neurobiology, The Weizmann Institute of Science, Rehovot, Israel michal.schwartz@weizmann.ac.il Several years ago we proposed, contrary to the prevailing wisdom, that maintenance and repair of the central nervous system (CNS) needs the assistance of the immune system. Because both innate and adaptive immunity were known to be implicated in the pathologies of inflammatory and non-inflammatory neurodegenerative diseases (such as multiple sclerosis in the first instance and Alzheimer's disease in the second), the question arose: under what conditions does immune activity operate as part of the repair process and thus need to be boosted, and when does it become part of the pathology and hence require modulation? According to this concept, T cells that recognize CNSrelated autoantigens ("autoimmune" T cells) protect neural tissue and induce neural cell renewal from endogenous stem cells by locally providing growth factors and causing microglia to adopt supportive phenotypes. Using an animal model of Alzheimer's disease, we demonstrated that T-cell-based vaccination with a weak agonist of autoantigens can shift the default microglial phenotype towards one that enables microglia to engulf aggregated β-amyloid and produce growth factors, leading to arrest of disease progression and induction of functional rescue, tissue repair, and even neurogenesis. A similar T cellbased approach was shown by our group to be applicable in acute brain injury, stroke, and other neurodegenerative conditions. Our studies argue in favor of autoimmunity as a physiological system of defense and maintenance against the neurodegenerative diseases or age-related cognitive losses that result, at least in part, from aging of the immune system. Jonathan Kipnis Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska 68198-5800, USA Recent results obtained in rodents suggest that T cells have an important role in several aspects of neuronal plasticity, such as hippocampal neurogenesis and spatial learning and memory. Cognitive performance, tested in learning and memory tasks, was impaired in immune-deficient mice compared to their wild-type counterparts. Superior performance of wild type mice was correlated with activation of GFAP-positive astrocytes in the hippocampus. Restoration of a T cell pool in immune deficient mice, which resulted in improved cognitive abilities, was also accompanied by significant increase in GFAP positive cells in their hippocampi. Our in vitro results provide evidence that astrocytes, major non-neuronal cells that actively participate in neuronal networking, activated with T cells possess a phenotype supportive of neuronal functioning and establishment of neuronal network. Therefore, T cell-mediated improvement of cognitive functions might encounter, at least in part, on an enhanced neuronal plasticity supported by astrocytes as a result of interaction with T cells. Based on this hypothesis, we boosted a T cell response by immunization with a synthetic Copolymer-1 (glatiramer acetate), which gives rise to T cells with partial recognition of brain-specific antigens. Such immunization significantly improved the performance of wild type mice in cognitive task. These findings suggest that the decline in immune activity known to be associated with several mental and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease, might account, at least in part, for the cognitive dysfunction observed in these patients. Such new understandings could lead to the identification of new therapeutic targets and the development of new therapeutic approaches.Demyelinating lesions or "plaques" in multiple sclerosis (MS) have distinct histological features that reflect their stage of evolution. To identify key molecules involved at different stages during lesion progression, we performed proteomic profiling of three types of MS plaques using mass spectrometry and bio-informatics. Fresh frozen autopsy brain samples from six MS patients and age-matched controls were classified as (1) acute plaque (AP, characterized by presence of inflammatory cells, ongoing demyelination), (2) chronic active plaque (CAP, characterized by chronic demyelination, astrogliosis with active rim) or (3) chronic plaque (CP, characterized by lack of inflammatory cells, dense astrogliosis). Samples from the plaques were isolated by laser capture microdissection and global protein identification was carried out by SDS-PAGE, in-gel protease digestion, liquid chromatography and mass spectrometry. Identified proteins were analyzed by bio-informatics software (SEQUEST, INTERACT, INTERSECT and emPAI) for identification of unique proteins and quantitation. A total of 3894 proteins were identified. There were 77 proteins associated with active inflammation and 102 unique to chronic lesions. These results provide the first and most comprehensive information on global protein expression in MS plaques, enhance our understanding of the molecular pathogenesis of MS lesions and identify potential key molecules involved in disease progression that offer possibilities for therapeutic targets. CS01-04 Endogenous neuroprotective mechanisms in multiple sclerosis T. Zeis a , U. Graunman a , A.J. Steck a , W. Brück b and N. Schaeren-Wiemers a a Neurobiology, Department of Research, University Hospital Basel, Pharmacenter, Switzerland; b Institute of Neuropathology, Georg-August-University Göttingen, Germany MS is a chronic inflammatory disease of the CNS leading to focal destruction of myelin. However, the earliest changes that lead to lesion formation are not known. Our recent microarray analysis of normalappearing white matter (NAWM) revealed upregulation of a number of functionally related genes known to be involved in endogenous neuroprotection as well as in maintenance of cellular homeostasis (Brain Pathology 2003) . The question arises, which of these alterations occur in the early stage of MS, and which are the consequences after long disease progression. Biopsy tissues are routinely embedded in paraffin, and therefore, we have established a protocol for successful isolation of total RNA allowing quantitative RT-PCR analysis, and detection of low abundant genes. In an ongoing study, we have characterized NAWM from a needle biopsy of an 18-year old female patient. Immunopathological examination revealed pathology, which is in agreement with Pattern III lesions after the classification by Lucchinetti and Brück (2002) . We isolated total RNA from NAWM tissue samples of this patient and performed qRT-PCR. Strikingly, we could detect very high NOS expression levels in NAWM of this biopsy, which was much less evident in NAWM of post-mortem tissues. Based on these data, we believe that these alterations reflect very early changes in MS. We speculate that depending on the regulation of the endogenous neuroprotective mechanisms, lesion formation might either be prevented or sustained. CS02-01 Hormonal effects on autoimmune disease C.C. Gienapp, F. Song and T. Shawler The Ohio State University, Columbus, Ohio, USA Women with multiple sclerosis (MS) experience a decreased relapse rate during late pregnancy, with a flare of disease 3-6 months postpartum. We examined the effect of pregnancy on experimental autoimmune encephalomyelitis (EAE) in the SJL mouse and the impact of pregnancy on ongoing disease. Pregnant animals showed a reduced incidence of EAE and a decrease in clinical severity, while mice induced for EAE during the post-partum period showed more severe disease. The most striking results were in mice who became pregnant after the acute disease period, showing complete protection during mid and late pregnancy. Cells from protected mice produced less TNF-α and more IL-10 than non-pregnant controls. CD4/CD25 + cells were not increased in mice immunized during pregnancy and the primary source of IL-10 was CD11b + cells. We explored the role of dendritic cells (DC) in mediating pregnancy-induced protection from EAE. Similar increases in costimulatory molecules, PDL1 and PDL2, were noted on DCs from mice treated with estriol, the pregnancy estrogen. Transfer of DCs from estrioltreated mice protected recipients from EAE. CS02-02 The intricate interface between immune and metabolic regulation: A role for leptin in the pathogenesis of multiple sclerosis? Giuseppe Matarese Istituto di Endocrinologia e Oncologia Sperimentale, Consiglio Nazionale delle Ricerche, via S. Pansini 5 -80131 -Napoli, Italy; email: gmatarese@napoli.com Over the last few years a series of molecules known to play a function in the metabolic function have also been shown to play an important role in the regulation of the immune response. In this context, the adipocytederived hormone leptin has been shown to regulate the immune response both in normal as well as in pathological conditions. genetic leptin deficiency, anorexia nervosa, malnutrition) are associated with increased susceptibility to infections. Conversely, immune-mediated disorders such as autoimmune disorders are associated with increased secretion of leptin and production of pro-inflammatory pathogenic cytokines. Leptin could represent the "missing link" between immune response, metabolic function and nutritional status. Indeed more recently, leptin-deficient mice have been shown to be resistant to a series of experimentally-induced autoimmune disorders including experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Normal wild-type mice show increased secretion of leptin in serum upon EAE induction, and brain inflammatory infiltrates stain positive for leptin. Finally, leptin neutralization with anti-leptin antibodies, improves EAE course and progression and profoundly alters intracellular signalling of myelin-reactive T cells. These data suggest that leptin can be considered as a major link between immune and metabolic regulation and that strategies aimed at interfering with the leptin axis could represent innovative therapeutic tools for autoimmune disorders. CS02-03 The dual role of the sympathetic nervous system in chronic inflammation R. H. Straub Laboratory of Neuroendocrino-Immunology, Department of Internal Medicine I, University Hospital Regensburg, GermanyIn this presentation, we demonstrate the dual pro-and anti-inflammatory role of the SNS (SNS) in inflammatory joint disease via distinct adrenoceptors. The dual role of the SNS depends on involved compartments, on timing of distinct effector mechanisms during the inflammatory process, on availability of respective adrenoceptors on target cells, and on an intricate shift from beta-to alpha-adrenergic signaling in the progressing course of the inflammatory disease (beta-to alpha-adrenergic shift). A further critical point for the dual role of the SNS in inflammation is the underlying change of immune effector mechanisms during the process of disease progression and also the behavior of sympathetic nerve fibers in inflamed tissue (nerve fiber loss). In quintessence, in very early stages of inflammatory joint disease, the SNS plays a predominant proinflammatory role whereas in late stages of the disease the SNS most probably exerts anti-inflammatory effects. Since patients with rheumatoid arthritis most often present themselves in the chronic phase of the disease, support of anti-inflammatory sympathetic pathways can be a promising therapeutic option in chronic inflammatory diseases. CS02-04 Experimental stroke causes early immune activation followed by systemic immunosuppression H. Offner Oregon Health and Science University, Portland, OR, USA Induction of stroke not only produces local ischemia and brain damage, but also has profound effects on peripheral immune responses. Our general hypothesis is that focal cerebral ischemia alters immunocyte function through brain-to-spleen signaling via the sympathetic nervous system. We observed an initial rapid intra-splenic activation of immunocytes, including T-lymphocytes. Some of these T-cells appeared to be released from the "post-stroke" spleen within hours, homed to the brain and secreted cytokines that amplified peri-infarct inflammation. Subsequently, there was loss of immune competence, characterized by splenic atrophy and increased splenocyte apoptosis. This was accompanied by reduced T cell proliferation and secretion of inflammatory cytokines. These changes produced a drastic reduction in B cell numbers, a novel increase in CD4 + FoxP3 + regulatory Tcells, and an increase in the percentage of non-apoptotic macrophages/ monocytes. Immunosuppression in response to brain injury may account for the reduction of inflammatory factors in the stroke affected brain, but also potentially could curtail protective immune responses in the periphery. These findings provide new evidence to support the contention that damage to the brain caused by cerebral ischemia produces immunosuppression caused by cell death as well as an increased presence of CD4 + FoxP3 + Treg cells. The net effect for a stroke survivor would be enhanced risk for crippling systemic infections and septicemia. CS03-01 Clinical characteristics and pathophysiology of the biopsied neuromuscular junction in Japanese MuSK antibody-positive myasthenic patients Investigation of the unique susceptibility of extraocular muscle (EOM) by myasthenia gravis (MG) has led to novel insights into the disease and neuromuscular transmission. The reasons for their susceptibility are multiple, based on the molecular and physiological properties of EOM, the requirements of the ocular motor system, and the nature of the autoimmune pathology. Even minor EOM weakness will sufficiently misalign the visual axes to produce dramatic symptoms and proprioceptive feedback is limited to overcome such a deficit. EOM synapses have structural features that would make them susceptible to neuromuscular blockade; secondary synaptic folds are simplified and certain fibers contract in a tonic fashion such that any compromise of transmission would lead to a reduction contraction force. The combined features of high neuronal stimulation and junctional features that make transmission failure more likely suggests these neuromuscular junctions behave more like central nervous system synapses. EOM junctions have lower levels of intrinsic complement regulatory factors, which would make them more prone to damage by acetylcholine receptor (AChR) antibodies. Among ocular MG patients, serum AChR antibody levels are low, which would support the concept that EOM junctions are more susceptible to antibody injury than other junctions. Specific inhibition of the complement system could be of particular utility in treatment of ocular myasthenia. Improvement in understanding of the EOM susceptibility to MG may continue to provide insights into MG pathogenesis and neuromuscular transmission. We have focussed on 'hyperplastic' and neoplastic changes in the MG thymus. Patients with early-onset MG (EOMG; age < 40) or thymomas have pathogenic antibodies against the native muscle acetylcholine receptor (AChR). Autoantibodies have not yet been detected in standard assays (against AChR or MuSK) in 5-10% of patients with typical generalised MG ('anti-AChR − '), despite their mild thymic hyperplasia.In >80% of EOMG patients, infiltrates of T-cell areas and germinal centres (GC) compress the hyperplastic medullary thymic epithelial cells (mTEC). Some mTEC express HLA-class II and unfolded AChR subunits, and could prime helper T-cells (Th) against AChR in the abnormal adjacent professional lymphoid tissue.We hypothesise that: (1) the primed Th then evoke 'early autoantibodies'; these attack nearby thymic myoid cells expressing intact AChRwhich frequently label for activated C3c and even C9 complement components. The similar labelling in >50% of the anti-AChR − patients suggests that they too belong to the EOMG spectrum; (2) autoantibodies diversify in the GC to recognise native AChR, including the fetal isoform expressed by myoid cells.Thymomas are neoplasms of TEC, which again express isolated AChR subunits. These latter increase when thymomas recur or metastasise; they are also produced spontaneously by mature plasma cells in the thymomas, implying stimulation by some cell type(s) there. CS03-04 Antibodies to presynaptic receptors acting for synaptic plasticity in myasthenias M. Takamori and K. Otsuka Neurological Center, Kanazawa-Nishi Hospital and Kanazawa University, Japan In myasthenia gravis (MG), acetylcholine (ACh) release from the nerve terminal is upregulated when synaptic transmission is postsynaptically impaired by anti-acetylcholine receptor (AChR) antibodies. The mechanism of this synaptic plasticity acts via intraterminal second messengers. This depends on a cytoplasmic free Ca 2+ -dependent components, but is not caused via regulatory effects on the presynaptic voltage-gated Ca 2+ channels (VGCCs). Independently of membrane depolarization, the presynaptic Ca 2+ influx is triggered via the phospholipase C activation and phosphatidylinositide (PI) pathway which is not affected by intracellular Ca 2+ stores. The one is the membraneassociated Ca 2+ influx channels, transient receptor potential classical subfamily-3 (TRPC3), which is activated through the pathway including brain-derived neurotrophic factor and receptor tyrosine kinase B. The other is the G-protein-coupled M1 muscarinic AChR (M1 mAChR), which is efficient to P/Q-type VGCC-mediated Ca 2+ influx via the PI pathway and, in a different manner, physiologically associated with TRPC-mediated Ca 2+ influx via the activation by diacylglycerol. Forty MG patients, 26 positive and 14 negative for anti-AChR antibodies, were studied for antibodies to TRPC3 peptide Bone marrow stromal cells (MSCs) have the capability under specific conditions to differentiate into various cell types. Here we demonstrate a highly efficient and specific induction of Schwann cells (peripheral glial cells), neurons and skeletal muscle cells from both rat and human MSCs.Schwann cells, which constitute peripheral nervous system, are known to support axonal elongation. MSCs were treated with beta-mercaptoethanol followed by retinoic acid (RA) and cytokines expressed markers of Schwann cells. These cells promoted axonal regeneration, and resulted in functional recovery when transplanted into the spinal cord injury model. Neuronal induction could be achieved by NICD transfection followed by trophic factor administration of bFGF + CNTF + forskolin. MSCs expressed markers related to neural stem cells after transfection with NICD, and subsequent administration of trophic factors induced post-mitotic neurons without differentiation of glial cells. Some of the induced cells showed action potentials compatible with characteristics of functional neurons. Further treatment of the neurons with GDNF increased the proportion of dopamineproducing cells and transplantation of these neurons showed improvement in rat model of Parkinson's disease. Highly efficient skeletal muscle induction could also be achieved by the administration of a combination of cytokines followed by NICD transfection. Induced cells differentiate into muscle fibers upon transplantation into degenerated muscles. Since induced cells contained satellite-like cells, they contributed to subsequent regeneration upon repetitive damage without additional transplantation of cells. Our MSC differentiation system may contribute substantially to a major advance toward eventual cell-based therapies for neurodegenerative and muscle degenerative diseases. CS05-04 Clearance of tissue debris by TREM2-transduced myeloid cells promotes recovery of experimental autoimmune encephalomyelitis Kazuya Takahashi 1 , Marco Prinz 2 , and Harald Neumann 1 1 Neural Regeneration Unit, Institute of Reconstructive Neurobiology, University Bonn LIFE and BRAIN Center and Hertie-Foundation, Sigmund-Freud-Str. 25, 53127 Bonn, Germany; 2 Department of Neuropathology, University Hospital Göttingen, 37075 Göttingen, GermanyBackground: Inflammation can successfully be prevented in multiple sclerosis, but tissue repair is still a major challenge for therapy. Bone marrow or blood-derived hematopoietic myeloid precursors are suitable and accessible sources for cell-based repair therapies. Efficient removal of apoptotic cells and cellular debris without inflammation during degenerative central nervous system (CNS) diseases is essential for repair of the injured tissue and disease recovery. Methods: Bone marrow-derived myeloid precursors were expanded in culture and lentivirally transduced with the microglial innate immune receptor triggering receptor expressed on myeloid cells-2 (TREM2). TREM2-transduced myeloid cells were intravenously applied in mice afflicted by experimental autoimmune encephalomyelitis (EAE). Results: TREM2-transduced myeloid precursors showed increased phagocytic activity and anti-inflammatory cytokine production after stimulation. Intravenous application of TREM2-transduced myeloid cells at the peak of EAE led to a significant amelioration of clinical symptoms, reduction in axonal damage and prevention of demyelination. The TREM2-transduced myeloid cells migrated into the inflammatory spinal cord lesions within a few hours after i.v. application and locally created an anti-inflammatory cytokine milieu within the spinal cord. The TREM2 expressing myeloid cells resolved inflammation by removing cellular debris and creating a milieu supportive for repair of injured tissue. Furthermore, we identified TREM2 as a novel target for the therapy of inflammatory autoimmune brain diseases. Bernhard Hemmer Neuroimmunology Group, Department of Neurology, Heinrich Heine-University, Düsseldorf, GermanyMultiple sclerosis (MS) is a chronic disease of the CNS that is characterized by inflammation, demyelination and axonal injury. Although the etiology of MS is still unknown, many findings point to a central role for the immune system in the pathogenesis of the disease. The occurrence of local antibody synthesis in CSF demonstrated by the presence oligoclonal bands or intrathecal IgG production is still the only diagnostic laboratory marker in MS. Paradoxically, however, B cells were neglected in MS research for decades owing to their dispensable role in EAE and the lack of suitable technology, and it is only recently that their impact in MS pathogenesis has been thoroughly studied. B cells from MS lesions or CSF comprise dominant clonotypes containing replacement mutations in their Bcell receptor genes, compatible with an antigen-driven selection process. The same B-cell clonotypes are found in the CSF during the course of the disease, implying that they are periodically recruited, or that they persist in the CNS compartment. This model is compatible with other CSF parameters. B-cell cytokines such as BAFF are also detected in MS lesions. Plasma blasts and plasma cellsterminally differentiated B cells that are usually only found in acute infectious diseasescan be identified in the CSF compartment of MS patients. Their number correlates not only with local IgG synthesis, but also with the extent of CNS inflammation. Although these findings strongly support a role for the humoral immune response in MS, the specificity and function of this response remains to be determined. CS06-02 Immunomodulation by statins and their potential role in MS therapy Scott S. Zamvil Department of Neurology and Program in Immunology, University of California, San Francisco, San Francisco, CA, USA Statins, which are widely prescribed for cholesterol reduction, have immunomodulatory properties that may be beneficial in treatment of MS and other Th1 mediated autoimmune diseases. Data indicate that immunomodulation by statins occurs in a cholesterol-independent manner. Recently, we have connected isoprenoid intermediates of the mevalonate pathway with signaling pathways in Th1/2 cell differentiation. In vivo depletion of the isoprenoid intermediates geranylgeranyl-pyrophosphate (GGPP) and farnesyl-PP by oral atorvastatin inhibited Th1 responses by decreasing geranylgeranylated RhoA and farnesylated Ras at the plasma membrane, which was associated with reduced phosphorylation of ERK and p38, and reduced DNA binding of their co-target, c-fos. Our recent data indicate that oral atorvastatin and rosuvastatin are highly potent in inducing Th2 differentiation by inhibiting ERK signaling. However, while oral simvastatin inhibits T cell proliferation, it is less effective in inducing Th1/2 modulation. These individual differences may be important when choosing a statin for testing in human autoimmune conditions. Based primarily upon the beneficial immune modulation of atorvastatin in EAE, we are conducting a multi-center, randomized, placebocontrolled study to determine whether oral atorvastin (Lipitor®) (80 mg) will reduce the risk of conversion to clinically definite MS in patients that have experienced a clinically isolated syndrome (CIS). This trial, which is funded by the Immune Tolerance Network (ITN) of the NIH is actively recruiting patients. Recruitment of regulatory T-cells to treat EAE Ralf Gold 1 , Shin-Young Na 2 , Fred Lühder 1 , Thomas Hünig 2 1 Institute for MS Research, University of Göttingen, D-37073 Göttingen, Germany; 2 Institute for Virology and Immunobiology, University of Würzburg, Germany CD4 + CD25 + regulatory T cells (T reg cells) play a key role in controlling autoimmunity and inflammation. Therefore, therapeutic agents capable of elevating numbers and/or increasing effector functions of this T cell subset are highly desirable. In a previous report we showed that a superagonistic monoclonal antibody specific for rat-CD28 (JJ316) expands and activates FoxP3 + T reg cells in vivo. Single intravenous administration of a low dose of the CD28 superagonist into either DA rats or Lewis rats suffering from experimental autoimmune encephalomyelitis (EAE) proved to be highly efficacious.In further experiments a novel, spontaneous mouse EAE mediated by CD8 − T cells was treated with T reg cells. In transgenic mice expressing the model antigen ovalbumin (OVA) in oligodendrocytes (ODC), CD8, but not CD4 T-cells with transgenic OVA-specific T-cell receptors (TCR) caused fulminant EAE. Spontaneous development of severe EAE mainly affected cerebellum and brainstem, suggesting the name 'Charcot mouse' for this specific phenotype. Transfer of polyclonal T reg cells activated by a novel mouse CD28-specific superagonistic monoclonal antibody (D665) protected all animals from lethal EAE. In surviving mice, transferred regulatory T-cells were found in the affected regions of the CNS. brain and spinal cord.Our data indicate that polyclonal T reg cells have therapeutic efficacy in CD4 and CD8 mediated rodent EAE. Immunization with irradiated autologous autoreactive T cells (T cell vaccination) is shown to induce regulatory T cell responses in patients with multiple sclerosis (MS) . The regulatory T cell reactivity is thought to contribute to suppression and depletion of circulating myelin-reactive T cells used for vaccination. Here, the characteristics and regulatory mechanism of CD8 + anti-idiotypic T cells and CD4 + regulatory T cells induced by T cell vaccination will be reviewed, with an emphasis on newly characterized CD4 + regulatory T cells (PNAS, 103, 5024-5029, 2006) . These CD4 + regulatory T cell lines were generated from patients with MS that received immunization with irradiated autologous myelin basic protein (MBP)-reactive T cells. CD4 + regulatory T cell lines had marked inhibition on autologous MBP-reactive T cells and displayed two distinctive patterns distinguishable by the expression of transcription factor Foxp3 and cytokine profile. The majority of the T cell lines had high Foxp3 expression and secreted both interferon-γ and IL-10 as compared to the other pattern characteristic of low Foxp3 expression and predominant production of IL-10 but not interferon-γ. CD4 + regulatory T cell lines of both patterns expressed CD25 and reacted with activated autologous T cells but not resting T cells, irrespective of antigen specificity of the target T cells. It was evident that they recognized preferentially a synthetic peptide corresponding to residues 61-73 of the IL-2 receptor alpha-chain. T cell vaccination correlated with increased Foxp3 expression and T cell reactivity to peptide 61-73. The findings have important implication in the understanding of the role of CD4 + regulatory T cell response induced by T cell vaccination. CS07-01 Innate signaling for CNS glial responses Trevor Owens Medical Biotechnology Center, University of Southern Denmark, Odense, Denmark Signals from both the central nervous system (CNS) and immune system contribute to pathology in inflammatory demyelinating diseases such as multiple sclerosis. We study innate signals for glial response, that are both exogenouslyand endogenously-derived. This presentation will describe the activation of glial cells in the mouse brain, in response to axonal injury, cuprizone demyelination and cytokine-expressing adenovirus. Glial activation in response to these experimental stimuli is initiated independently of immune involvement, and results in chemokine and cytokine secretion. Injury-induced microglial Toll-like receptor-2 signals act as upstream on-switches for glial cytokine and chemokine responses, and microglial proliferative expansion. Whereas injury-or cuprizone demyelination-induced glial cytokines and chemokines appear sufficient for entry of macrophages and T cells to the CNS, leukocyte entry in response to transgenic or adenoviral interferon gamma-induced chemokine production requires additional signals, that can be provided through recognition of the pathogen-derived innate trigger, pertussis toxin. Glial cross-talk is mediated by cytokines, with characteristic intracellular signals. Activated microglia and macrophages show antigen-presenting cell surface phenotypes, and can activate T cells in culture. However, the T cells that enter the CNS, in the absence of deliberate immunization, require signals additional to chemokines and cytokines for functional activation. Our findings identify a complex of signaling events that must combine for immune pathology in the CNS. CS07-02 Toll-like receptor 3 on astrocytes mediates neuroprotective responses and can be activated by an endogenous protein agonist Toll-like receptors (TLRs) are expressed by a variety of cell types and function as sensors of microbial structures such as endotoxins, peptidoglycans and nucleic acids. In response to such molecules TLRs generally activate pro-inflammatory host defense responses. Human astrocytes express a limited repertoire of TLRs that are only found on the cell surface.In particular TLR3 appears to be special to astrocytes since it is strongly and preferentially upregulated in response to a variety of stimuli including cytokines, oxidative stress and TLR agonists themselves.Gene profiling of the response of astrocytes to TLR3 activation revealed a powerful response dominated by anti-inflammatory cytokines, neuroprotective molecules, remyelination signals and several chemokines. Given this apparent tissue repair function of surfaceexposed TLR3 on astrocytes we examined the possible presence of an endogenous TLR3 agonist in the central nervous system. Indeed, genomescale screening of a brain tumor-derived cDNA bank revealed a protein that is preferentially expressed in the human CNS and can activate a TLR3-dependent response by astrocytes. CS07-03 Dendritic cells and lymphoid microenvironments in the inflamed CNS Francesca Aloisi 1 , Barbara Serafini 1 , Roberta Magliozzi 1,2 , Abhilash Vora, Richard Reynolds 2 1 Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Rome, Italy; 2 Department of Cellular and Molecular Neuroscience, Imperial College London, London, UK Chronic inflammatory diseases, such as autoimmune and infectious diseases, are associated with repeated recruitment and persistence of immune cells in the target tissue. Evidence is accumulating that, even within an immune privileged site such as the central nervous system (CNS), chronic inflammation leads to the organization of lymphoid microenvironments that may contribute to sustain a local immune response against microbial or self antigens. Dendritic cells (DC) recruited to the inflamed CNS are thought to play a key role in this process. In multiple sclerosis and its animal model EAE, the perivascular space of inflamed blood vessels in the white matter is the preferential site where dendritic cell accumulate, take up myelin components and interact with CNS-infiltrating lymphocytes. The inflamed meninges provide an even more supportive environment for the generation of ectopic lymphoid-like tissue. Here, myeloid and plasmacytoid DC can be detected and B-cell follicles with follicular dendritic cells (FDC) develop in a substantial proportion of patients with secondary progressive MS. FDC, which are of mesenchymal origin, have a central role in the selection of memory B cells during germinal center reactions. Remarkably, development of ectopic lymphoid tissue in MS is associated with extensive cortical demyelination and an aggressive clinical course. It is proposed that 'seeding' of the brain by lymphocytes and antigen presenting cells and their organization in lymphoid-like structures exacerbates the inflammatory process as a consequence of an increased availability of pathogenic, presumably autoreactive, lymphocytes and their products. Prevention or eradication of lymphoid microenvironments nested within the CNS should be considered as an important goal for therapeutic intervention. CS07-04 Innate immunity and bone marrow stem cells in brain diseases and repair receptors (TLR), cytokines, chemokines and proteins of the complement system in the circumventricular organs and other structures devoid of blood-brain barrier, diffuses progressively into microglia across the brain parenchyma and may lead to an adaptive immune response. The cerebral innate immunity is likely to be an essential player in the etiology of inflammatory CNS disorders resulting from infection as well as those assumed to have an immune etiology. On the other hand, molecules of the innate immunity have been found to trigger the production of neurotrophic factors and promote neurorepair and remyelination in response to brain injuries, trauma and toxin-induced demyelination.Exciting new data will also be presented in regard to the role of newly recruited microglia from the bone marrow stem cells in CNS diseases, such as Alzheimer's Disease (AD). This disease is characterized by the deposition of aggregated beta-amyloids that form amyloid plaques. These senile plaques are composed of microglia, which infiltrate the core of amyloid bodies and these cells have different origins. Those deriving from the bone marrow stem cells have the ability to eliminate the plaques and inhibition of this process was found to aggravate the disease. These data provide novel insights for the development of new therapeutic strategies using bone marrow stem cells as vehicle for gene therapy and the elimination of toxic senile plaques.Our work on this subject is currently funded by the Canadian Institutes of Health Research (CIHR). CS08-01 Going solo or in need of a friend: Ganglioside and lipid clusters form novel antigenic determinations in GBS S. Kusunoki Kinki Univeristy School of Medicine, Osaka, JapanAnti-ganglioside antibody titers are frequently elevated in the acute phase sera from patients with Guillain-Barré syndrome (GBS). They are useful diagnostic markers as well as possible pathogenetic factors of GBS. Antibody assay has been usually performed by ELISA, in which each purified ganglioside is used as an antigen. However, because each ganglioside is present in the plasma membrane, where it is colocalized with phospholipids and other gangliosides to form lipid rafts, we should pay attention on the effects of such other lipid molecules. We recently performed ELISA by the use of a mixture antigen composed of a ganglioside and a phospholipid. As a result, anti-GM1-positive sera had higher antibody titers against a mixture of GM1 and several phospholipids as phosphatidic acid than against GM1 alone. In contrast, no phospholipid provided significant enhancement on the anti-GQ1b antibody titers. Sphingomyelin provided decrease of the activity for both anti-GM1 and Anti-GQ1b antibodies. We next used a mixture antigen composed of two different gangliosides and found that some GBS sera had antibodies specific for a ganglioside complex (GSC), which is a conformational epitope formed by two gangliosides such as GM1 and GD1a (GM1/GD1a), GD1a/GD1b, GD1b/GT1b and GM1/GT1b. Anti-GSC-positive GBS were featured by antecedent gastrointestinal infection and lower cranial nerve deficits. Among the anti-GSC antibodies, anti-GD1a/GD1b and/or anti-GD1b/GT1b antibodies were associated with severe disability and a requirement of mechanical ventilation. They may be predictors of severe GBS. We should consider clustered antigenic epitopes to determine the pathogenetic role of antiganglioside antibodies in GBS. CS08-02 It's all in the genes: Campylobacter polymorphisms direct the clinical features of GBS N. Yuki Department of Neurology, Dokkyo Medical University School of Medicine, Tochigi, Japan Ganglioside mimicry of Campylobacter jejuni lipo-oligosaccharide (LOS) is a cause of Guillain-Barré syndrome (GBS). GM1-like and GD1alike LOSs are synthesized by Campylobacter sialyltransferase Cst-II, Nacetylgalactosaminyl-transferase CgtA, and galactosyltransferase CgtB. C. jejuni is grouped into several LOS locus classes based on the organization of the LOS biosynthesis genes. The fifty-first of the amino acid determines its enzymatic activity. Cst-II (Thr51) can make GM1-like and GD1a-like LOSs, whereas Cst-II (Asn51) can make GT1a-like LOS. Neuropathic strains with Cst-II (Thr51) expressed the GM1 and GD1a epitopes, whereas the strains with Cst-II (Thr51) expressed the GQ1b epitope. Patients infected with Cst-II (Asn51) strains had anti-GQ1b antibodies, and the patients showed ophthalmoplegia and ataxia. Then the patients develop ophthalmoplegia and ataxia. In other words, the Cst-II polymorphism determines the clinical presentation, GBS or Fisher syndrome. Now we have proposed a new paradigm that microbial genetic polymorphism can determine the clinical presentation of human autoimmune disease. Onset can be rapid, leading to total paralysis within 48 h. The patients left severely disabled or dead represent a major social and economic burden. Recent clinical-serological studies have identified serum anti-ganglioside antibodies in GBS cases and chronic neuropathies. Gangliosides are sialic acid-containing glycosphingolipids highly enriched in the nervous system. Anti-GM1 and -GD1a antibodies characterise the motor axonal form of GBS and anti-GQ1b antibodies characterise Miller Fisher syndrome (MFS) . In murine models, we have shown that anti-ganglioside antibodies target gangliosides in neuronal and glial membranes, including motor nerve terminals. Morphologically, dense antibody and complement deposits are found in the synaptic cleft and on perisynaptic Schwann cells (pSCs), alongside a loss of neurofilament with destruction of the nerve terminal, and pSC death. The terminal attack complex of complement is critical to development of acute neural injury by allowing calcium ingress, activation of calpain and degradation of the nerve terminal. Human immunoglobulin therapy also protects against nerve injury through anti-idiotypic effects. These studies show that nerve terminal gangliosides can act as targets for human disease-associated autoantibodies and that this site provides a useful model system for testing new pathogenesis paradigms and therapeutic strategies. CS09-01 Regulating myelin basic protein-specific T cell responses in vivo A loss of tolerance in myelin-specific T cells is believed to contribute to the pathogenesis of multiple sclerosis. Our previous studies showed that CD4 + T cells specific for myelin basic protein (MBP)121-140, a highly immunogenic MBP epitope in B10.PL mice, are largely tolerized by clonal deletion in the thymus. However, the developmentally regulated expression of MBP allows MBP121-140-specific T cells to escape central tolerance in young mice when little MBP is synthesized. We defined the peripheral tolerance mechanisms that prevent these T cells from mediating autoimmunity later in life when they encounter MBP in the periphery. Regulatory T cells are essential to prevent MBP121-140-specific T cell mediated autoimmunity. Regulatory T cells do not prevent the proliferation, expansion or trafficking of MBP-specific T cells transferred into wild-type mice. Instead, Th1 cytokine production by MBP-specific T cells was strongly suppressed. Suppression of Th1 cytokines and protection from autoimmunity was abrogated if endogenous antigen-presenting cells (APCs) were activated during the first week after transfer of MBP-specific T cells into recipients. Interestingly, activating APCs thirty days after transfer of the MBP-specific T cells no longer triggered autoimmunity. Instead, the MBPspecific T cells that persisted in the periphery of wild-type mice responded to this stimulus by further suppressing their Th1 cytokine responses. The tolerant phenotype of MBP-specific T cells persisting in wild-type mice was dependent on both T cell intrinsic and host factors, and did not represent a terminally differentiated state as re-transfer of the regulated MBP-specific T cells into T cell-deficient recipients caused autoimmune disease. CS09-02 Natural regulation of CNS autoimmune disease Institute for Immunology and Infection Research, University of Edinburgh, King's Buildings, Edinburgh, UK, EH9 3JT UK The development of new therapeutics for autoimmune disorders will benefit from an understanding of the natural regulatory mechanisms that can promote resolution of disease. With this in mind, it is notable the multiple sclerosis (MS) often follows a benign "relapsing remitting" course. We are investigating the regulation of the mouse model of MSexperimental autoimmune encephalomyelitis (EAE). The disease in C57BL/6 mice peaks in the second week after immunization with the CNS autoantigenic peptide, . A recovery phase follows, correlating with a specific accumulation of CD4 + CD25 + cells within the CNS. These CNS-derived Tregs preferentially produce IL-10, express FoxP3, are anergic, act as potent suppressors of responder CD25 − cells in vitro, and their transfer in vivo accelerates recovery from EAE in recipient mice. CD25 − depletion exacerbates disease, and delays recovery. Moreover, CD25 − depletion after recovery also renders mice fully susceptible to re-induction of EAE. Data will be presented relating to origins, mode of action and specificity of the CNS Treg cells. Our data highlight the key role of Tregs in the natural recovery from an ongoing inflammatory autoimmune disease and in subsequent resistance, and that their effects appear to be within the target organ. Thus there is real potential for Treg-directed therapies to boost natural regulation of active autoimmune disease. CS09-03 Human natural Tregs are composed of two functionally distinct subsets of cells based on expression of MHC Class II ex vivo C. M. Baecher-Allan, C. Ashley, C. Costantino, G. Beriou, and H. A. HaflerHarvard Medical School and Brigham and Women's Hospital, Boston, MA, USA Natural CD25 high regulatory (Treg) populations, isolated from patients with multiple sclerosis (MS), have been shown to be decreased in their suppressive ability as compared to Tregs isolated from healthy donors. In these studies, the Tregs that express HLA DR (DR + Tregs) exhibit a strong and swift suppression while the DR neg Tregs induce a delayed inhibition accompanied by suppression of Th1 but increases in Th2 cytokines. The DR + and DR neg Tregs also exhibit striking differences in their production and utilization of IL-10. While both DR + and DR neg Tregs express high levels of FoxP3 and exhibit a strong cell contact-dependent suppression, DR neg Tregs can also suppress via the production of IL-10, which is not produced by the DR + Tregs. In contrast, the DR + Tregs express the highest levels of IL-10R, and target cell production of IL-10 inhibits DR + Treg regulatory function, resulting in increased co-culture proliferation. Whether IL-10 inhibition of DR + Treg suppression occurs by reducing the suppressive ability of the DR + Treg or by increasing the resistance of responder T cell to suppression is under investigation. Additional studies are also underway to assess the activity and IL-10 sensitivity of the individual DR + and DR neg Treg subsets isolated from patients with MS to elucidate the mechanism for the deficiency in suppression by the total Treg population when isolated from patients with MS. CS09-04 Foxp3-expressing regulatory T cells in self-tolerance and autoimmune disease Naturally occurring CD25 + CD4 + regulatory T cells (Treg) actively engage in the maintenance of immunologic self-tolerance and immunoregulation. They specifically express the transcription factor Foxp3 as a master control molecule for their development and function. Although several cell surface molecules have been reported as Treg-specific markers, such as CD25, GITR (glucocorticoid-induced TNF receptor family-related gene/protein), and CTLA-4, they are also expressed on activated T cells derived from CD25 − CD4 + naïve T cells. To identify Treg-specific molecules controlled by Foxp3, we performed DNA microarray analysis and found that several genes, including Gpr83, Ecm1, and helios, are specifically expressed by natural Tregs, some of which are Foxp3-dependent. CS10-01 Molecular mechanisms of T cell migration across the BBB in vivo Britta Engelhardt Theodor Kocher Institute, University of Bern, Bern, SwitzerlandIn multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (EAE), inflammatory cells migrate across the endothelial blood-brain barrier (BBB) and gain access to the central nervous system (CNS). In contrast, the involvement of the selectins, L-selectin, E-and P-selectin and their respective carbohydrate ligands such as PSGL-1 in this process has been controversially discussed. We have demonstrated that antibodies directed against E-and P-selectin or their major leukocyte ligand PSGL-1 do not interfere with the recruitment of inflammatory cells across the BBB or the development of clinical EAE. Taken together, our findings suggest that selectins are not required for leukocyte recruitment across the BBB during EAE and point to a predominant role of α4-integrins in this process. Our findings will be discussed in light of the potential risks versus benefits of anti-α4-integrin therapy of multiple sclerosis. CS10-02 Transmigration of human CD14 monocytes across the blood brain barrier induces their differentiation into dendritic cells Igal Ifergan and Alexandre Prat University of Montreal, Neuroimmunology Laboratory, CHUM-Notre-Dame Hospital, Montreal, Quebec, CanadaThe blood-brain barrier (BBB) plays an important role in regulating transmigration of immune cells into the central nervous system (CNS). Disruption of the BBB and trafficking of auto-reactive T cells and antigen presenting cells into the CNS are seen as important and early events in lesion development in Multiple Sclerosis (MS). However, to date there is still much controversy surrounding the presence and recruitment of dendritic cells (DCs) within the human brain. We elected to demonstrate that human peripheral blood CD14 monocytes cross the BBB and, by doing so, differentiate into a DC-like phenotype. Using our in vitro model of trans-human brain endothelial cells (HBECs) migration, we identified chemokine MCP-1 as the main chemoattractant for monocytes migration across the BBB. We further demonstrate that the presence of HBECs during the inflammatory process leads to differentiation of monocytes into immature (HLA-DR low , CD80 low , CD86 low and CD83 neg ) and mature (HLA-DR high , CD80 high , CD86 high , DC-SIGN high and CD83 high ) DC phenotypes. Such DCs have the capacity to initiate CD4 T cell immune responses. Immunohistochemistry studies performed on brain sections of MS patients also reveal the presence of CD83 high , HLA-DR high , CD123 high and DC-SIGN high expressing cells in acute lesions. Finally, we show that the in vitro differentiation of monocytes into DCs can be inhibited by blocking HBECs production of GM-CSF and TGF beta. Our data support the notion that unique populations of immature and mature DCs arise from migration of peripheral blood CD14 monocytes across the human BBB through the concerted actions of MCP-1, TGF beta and GM-CSF. CS10-03 Reactive oxygen species induce alterations in blood-brain barrier integrity Elga de Vries 1 , Gerty Schreibelt 1 , Gijs Kooij 1 , J. Hendriks 1 , Christien Dijkstra 1 , Eric Ronken 2In multiple sclerosis (MS), migration of monocytes across the bloodbrain barrier (BBB) is a crucial step in the formation of lesions in the central nervous system (CNS). To enter the CNS, monocytes have to cross the BBB, during which reactive oxygen species (ROS) play an essential role. Previously we showed in the animal model for MS, experimental allergic encephalomyelitis (EAE) that the anti-oxidant lipoic acid (LA) and the flavonoid luteolin suppressed the development of clinical signs and prevented new relapses, which was coupled to reduced cellular infiltration, axonal damage and oxidative stress.Our recent data demonstrate that monocyte adhesion to cultures of brain endothelium induces production of ROS by monocytes within minutes and monocyte adhesion to a model for the BBB in vitro leads to an enhanced permeability. Exogenously administered ROS specifically activates the signalling pathway of PI-3 kinase within the brain endothelium, subsequently inducing cytoskeleton and tight junction changes. Using life cell imaging techniques, we were able to visualize that superoxide in time induces bundles of F-actin and alterations in the integrity of the tight junction. Besides its modulating effect on the integrity of the brain endothelium, superoxide also induces specific alteration in brain endothelial gene and protein expression, revealing the upregulation of markers of oxidative stress and inflammation.Our results indicate that anti-oxidants may be suitable as a new therapy for MS not only to limit cellular infiltration into the brain but also by stabilization of the BBB to resist an inflammatory attack. CS10-04 Chemokines and BBB function CCL2), can also cause BBB 'opening' by inducing interendothelial junction protein redistribution and endothelial actin cytoskeleton rearrangement. Further examination revealed that endocytotic internalization of transmembrane tight junction proteins (occludin, claudin-5 and JAM-1) stimulated by CCL2 is important for disassembly of complex endothelial tight junctions as well as paracellular route formation. However, the fate of such internalized proteins is recycling back to the plasma membrane. Collectively, these data provide new information on the underlying mechanisms through which chemokines induce BBB disruption that is relevant to multiple disease states and hopefully, will provide insight about methods to preventing such a disruption. CS10-05 Effect of statins on lymphocyte migration across the bloodbrain barrier J. Greenwood Institute of Ophthalmology, London, UK Lymphocyte adhesion to brain endothelial cells (EC) is known to initiate a number of signaling cascades, the physiological significance of which remains poorly defined. However, signaling generated through engagement of CNS EC surface adhesion molecules, such as ICAM-1 and VCAM-1, or through an unidentified G-protein coupled receptor, has been shown to be essential for successful lymphocyte transendothelial migration. The identification of key components of these EC pathways raises the possibility that targeted pharmacological intervention may provide us with novel antiinflammatory strategies for the treatment of diseases such as multiple sclerosis (MS). Efficient transduction of ICAM-1 mediated signaling responses in CNS EC, and subsequent transendothelial migration of Tlymphocytes, is critically dependent on the activation of the small GTPase Rho. Pharmacological inhibition of Rho proteins can be achieved by preventing their posttranslational prenylation through inhibition of the cholesterol synthesis pathway that provides isoprenoid pyrophosphates. We have explored the effect of statins, potent inhibitors of the enzyme HMGCoA reductase which is the rate limiting step of cholesterol synthesis, upon ICAM-1-mediated signaling pathways and transendothelial lymphocyte migration in vitro and neuroinflammatory disease in vivo. We have shown that statin treatment of CNS EC inhibits lymphocyte migration and attenuates experimental CNS autoimmune disease. Data presented will further support the pleiotropic action of statins and their potential use in treating MS. CS11-01 Identification of a novel cytokine produced by encephalitogenic T cells and induced by IL-23 Burkhard Becher Division of Neuroimmunology, University of Zurich, Winterthurer Strasse 190 CH-8057 Zürich, SwitzerlandTissue-directed autoimmune diseases were widely held to be mediated by the conduct of self-reactive T H cells. We and others have previously shown that neither Th1 nor Th2 gene-expression serves as a reliable marker for encephalo-pathogenicity. In contrast to the APC-derived cytokine IL-12, IL-23 was shown to be absolutely critical for the induction of autoimmune disease. It becomes increasingly evident that IL-23 supports the expansion of a distinct lineage of CD4 + cells that produce IL-17 (Th IL-17 cells) and appear play a major role in autoimmune inflammation. Using high density transcriptomics, we could identify an additional gene specifically induced by IL-23 in memory T cells. We discovered an IL-10 related cytokine whose expression profile is identical with that of the disease associated IL-17. Its expression correlates exquisitely with that of IL17 pointing towards a major role of this illdescribed cytokine in inflammatory disease. Taken together, this molecule will help to define the encephalopathogenic phenotype of autoaggressive effector T H cells in the context of Neuroimmune disease. CS11-02 Chemokines in the nervous system: Beyond chemoattraction R.M. Ransohoff Neuroinflammation Research Center, Cleveland Clinic, Cleveland, OH, USA Inflammation of the central nervous system (CNS) entails the activation of resident microglia and recruitment of hematogenous leukocytes. Thus defined, inflammation accompanies most neurological disorders, including multiple sclerosis (MS), stroke, neoplasia, trauma and HIV-1-associated dementia, as well as Alzheimer's disease and other primary neurodegenerations. Chemokines comprise a family of peptides that act through G proteincoupled receptors (GPCRs) to regulate leukocyte migration throughout all tissues, in an exquisitely specific and flexible fashion. Initial studies asked how chemokines and chemokine receptors governed inflammatory cell recruitment to the CNS during immune-mediated or virus-induced inflammation. More recently, it has become clear that the CNS complement of constitutive chemokines supports developmental and neurophysiological functions as well as regulating the activation of microglia. Because GPCRs can serve as drug targets, these results have implications for the understanding and treatment of disease by neurologists.Supported by the US National Institutes of Health (NS38667; NS32151; NS54100), by the National Multiple Sclerosis Society (USA); by the Charles A. Dana Foundation; by the Robert Packard Center for ALS Research; and by the Nancy Davis Center Without Walls. CS11-03 Transferable tolerance by modified dendritic cells in experimental autoimmune encephalomyelitis CS11-04 Transcriptional regulation of the IL-23/IL-17 immune axis during CNS autoimmune inflammation T H 17 is a novel T cell subset driven by TGF-β, IL-6 and IL-23 that has potent pro-inflammatory activities. While TGF-β and IL-6 are sufficient for differentiation of naïve T cells into IL-17 producing cells, IL-23 is required for their expansion, survival and pro-encephalitogenic functions. The hypothesis that T H 17 is a distinct T cell lineage predicts that TGF-β and/or IL-6 must promote the expression of a transcription regulator that controls the immune repertoire of the IL-23/IL-17 immune axis. Using Affymetrix gene-expression analysis, we found a number of T H 17 cell-specific novel genes encoding putative proteins with potential DNA binding activities. Genetic depletion experiments confirmed that one of these DNA binding proteins, orphan nuclear receptor-like factor, is necessary for the differentiation and function of T H 17 cells. Mice lacking this orphan nuclear receptor are EAE resistant and deficient in T H 17 development. The identification of this transcription regulator provided further evidence that T H 17 is a distinct T cell lineage that has unique roles in immunity. The implications of these findings for IL-23/IL-17 immune axis regulation of CNS autoimmune inflammation will be discussed. We have been interested in defining the role of viral infections in autoimmunity.During my presentation I will discuss the following aspects:1. Our earlier studies have shown that viral infections can do both, enhance or abrogate an ongoing autoimmune process. The outcome depends on the timing of the infection in relation to the autoimmune process and the nature of the virus. In contrast, too strong cross-reactivities can lead to apoptosis of autoreactive lymphocytes and their demise and prevention of autoimmunity. Viral infections can be essential to create an inflammatory milieu to precipitate autoimmune diabetes. Our data indicate that the efficacy of Treg induction after virus infection depends on the epitope presented. In general, virally-induced Tregs are of poorer ability to prevent autoimmunity than Tregs induced to autoantigens. Autoantigen specific Tregs can be used in immunotherapy and act as bystander suppressors. CS12-02 Innate and adaptive immune requirements for the induction of CNS autoimmunity via virus-induced molecular mimicry [574] [575] [576] [577] [578] [579] [580] [581] [582] [583] [584] [585] [586] ) which shares 6/13 amino acids or mouse hepatitis virus papain-like proteinase (MHV 3821-3832 ) which shares only 3/13 amino acids with PLP 139-151 , develop an early-onset demyelinating disease mediated by PLP 139-151 -specific Th1 cells. Relative to the importance of innate immune signals in disease induction, we have found that disease is only induced when the mimics are delivered by genetically engineered TMEV, not by immunization with the synthetic peptide mimics in CFA. Proline at the secondary MHC class II contact residue is required for effective cross-reactivity as its addition to the native MHV sequence increases its ability to cross-activate PLP 139-151 -specific autoreactive T cells, while substitution of proline in the HI mimic peptide has the opposite effect. Early onset demyelinating disease and PLP 139-151 -specific cross-reactive responses are also induced by infection with viruses engineered to express 30-39 residues entirely encompassing the H. influenzae (HI39-TMEV) or MHV (MHV30-TMEV) epitopes indicating that the mimics are capable of being processed from their endogenous flanking residues. Our results thus describe a structural requirement for autoreactive T cell activation by potential PLP 139-151 mimic peptides, and provide further support for infection-induced molecular mimicry in the pathogenesis of autoimmune disease. CS12-03 Innate immune mechanisms that promote development of effector and regulatory CD4 lineages in EAE (Experimental Autoimmune Encephalomyelitis) We have previously shown that HSV-1 viral infection can provoke autoimmunity through interactions with both the innate and adaptive immune responses. Epitopes expressed by HSV-1 that cross-react with self antigen can trigger expansion of autoreactive T cells and consequent autoimmune disease. A second set of interactions between HSV-1 and the innate immune system also plays an essential role in effective expansion of pathogenic T cells. We will summarize our recent experimental analysis of interactions between viruses and the innate immune system that regulate development of autoimmune disease in EAE (Experimental Autoimmune Encephalomyelitis). These findings suggest that expression of the innate mediators Osteopontin (Opn) and TGF-β by dendritic cell subsets regulates Th17 and T-reg development and may play a key role in the development of EAE. Kirvan a a University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA; b National Institute of Mental Health, Bethesda, MD USA Molecular mimicry is the central theme of autoimmune sequelae following group A streptococcal infection in humans. Immune responses against the group A streptococcal bacterium are directed against host tissues and lead to the major manifestations of rheumatic fever including rheumatic heart disease and Sydenham chorea. Sydenham chorea, a movement disorder, is the major neurological manifestation of acute rheumatic fever. Human monoclonal antibodies were derived from Sydenham chorea and used to investigate the molecular mechanisms of immune pathogenesis.Human chorea monoclonal antibodies demonstrated specificity for streptococcal membranes and the group A streptococcal carbohydrate epitope, N-acetyl-beta-D-glucosamine, as well as the brain antigen lysoganglioside on the surface of neuronal cells in culture. Monoclonal autoantibodies (Mab), acute sera (IgG), and cerebrospinal fluid (IgG) recognized lysoganglioside and the intracellular protein tubulin which was identified as a 55 kd protein with the N terminal sequence MREIVHLQ. The nucleotide sequences and reactivities of the chorea mAbs were distinctly different from mAbs derived from rheumatic heart disease. When mixed with neuronal cells, chorea mAb 24.3.1, acute chorea serum IgG or CSF IgG demonstrated antibody-mediated cell signaling in neuronal cells by activation of calcium/calmodulin dependent protein kinase II activity and tritiated dopamine release. In summary, the data suggest mimicry and an antibody-mediated cell signaling mechanism in the brain which promotes dopamine release and chorea in humans. CS13-01 Effects of stress on immune function: The good, the bad and the beautiful F. S. Dhabhar Stanford University School of Medicine, Stanford, California, USA Stress is known to suppress immunity and increase susceptibility to infections and cancer. Paradoxically, stress exacerbates autoimmune diseases like multiple sclerosis, suggesting that stress may also enhance immune responses. Therefore, it is important to elucidate the conditions and mechanisms mediating these bidirectional effects of stress on immune function. It has been shown that acute stress experienced during primary antigen exposure increases memory T cell formation and induces a longlasting increase in immunity (International Immunology, 17:1059; American J Physiology, 289:R738). Acute stress experienced during antigen re-exposure enhances a secondary immune response (J Immunology, 156:2608). Stress-induced changes in leukocyte distribution (PNAS, 102:5808), epinephrine, and corticosterone (PNAS, 96:1059) , are systemic mediators, and gamma-interferon is one local mediator (PNAS, 97:2846) of immunoenhancement. In contrast, chronic stress is immunosuppressive (BBI, 11:286) . For example, chronic stress increases susceptibility to skin cancer by suppressing Type 1 cytokines and protective T cells while increasing suppressor T cell numbers (J National Cancer Institute, 97:1760) .We suggest that the adaptive function of a stress response is to promote survival, i.e., stress hormones prepare the cardiovascular, musculoskeletal and immune systems for oncoming challenges (e.g. attacker) detected by the brain. However, stress exacerbates disease when immunoenhancement is directed against innocuous or self antigens, or when immunosuppression/ dysregulation occurs during chronic stress. In view of the ubiquitous nature of stress and its significant immunoprotective and immunopathological effects, it is important to elucidate the mechanisms mediating stressimmune interactions and to meaningfully translate findings from bench to bedside. Coe and G. R. LubachHarlow Center for Biological Psychology, University of Wisconsin, Madison, WI, USA Background: The prenatal and early rearing environments play a critical role in initiating normal ontogeny. As a consequence, maternal disturbances can affect the maturational trajectory of the fetus and chronically affect functioning postpartum. Like the brain, the immature immune system is designed to 'learn' from the environment, which includes many aspects of maternal care. Methods: Pregnancy conditions of over 150 rhesus monkeys were manipulated, including by psychological disturbance of the gravid female, administration of dexamethasone, or viral infection. The impact on the infant's behavioral, immune and brain development was assessed. Results: Maternal stress, antenatal corticosteroids, and influenza exposure significantly affected neurological development. The offspring evinced immature neuromotor reflexes at birth, greater emotionality during the first year of life, and a smaller hippocampus as juveniles. Smaller hippocampal size was associated with a lower neurogenesis and altered pituitary-adrenal activity. Immune alterations were also evident, including a reduced capacity of neonatal lymphocytes to recognize 'self' from 'other 'antigen', decreased proliferative responses, and lower cytokine production in cultured cells. Several mediation pathways were identified including a predisposition for iron deficiency anemia in the fast-growing infant. In addition, prenatally stressed infants established a different profile of gut microbiota, with lower levels of protective Lactobacilli and Bifidobacteria. Conclusion: Adverse events during fetal life can affect health postpartum by impacting the brain and immune systems at vulnerable points and changing regulatory set points. In addition to maternal cortisol, disturbance of micronutrient transmission and establishment of an abnormal profile of gut bacteria were identified as important factors. CS13-03 Mechanism of interferon-induced depression: Involvement of hippocampal neurogenesis S. Kanba a , N. Kaneko b , K. Kudo c , K. Takemoto d and H. Wati d a Department of Neuropsychiatry, Kyushu University, Fukuoka, Japan; b Keio University, c Tokyo University, d Yamanashi UniversityThe therapeutic use of human interferon-alpha (hIFN-α) is known to cause various neuropsychiatric side effects. In particular, depression occurs in 30-45% of patients, frequently interrupting treatment. However, mechanisms underlying the depression caused by hIFN-α remain to be defined. Neurogenesis is known to be regulated by various cytokines. Therefore, we investigated the effect of subchronic hIFN-α treatment (5000-50,000 IU/kg/day) on neurogenesis in the adult rat dentate gyrus (DG). Immediately after a one-week treatment with hIFN-α, BrdU-labeled proliferating cells were decreased in the DG, but we did not detect an effect on cell differentiation into mature granule cells until three weeks after the treatment. After hIFN-α treatment, interleukin-1beta (IL-1β), a major proinflammatory cytokine, increased in the hippocampus. Co-administration of an IL-1 receptor antagonist (IL-1Ra) completely blocked the hIFN-α-induced suppression of cell proliferation in the DG. Our results demonstrated that hIFN-α suppressed cell proliferation but not cell differentiation in the DG, and that IL-1β plays an essential role in the suppression. The decreased cell proliferation caused by hIFN-α-induced IL-1β may be partly responsible for hIFN-α induced depression. CS13-04 Stress, immunity and cancer incidence Noriyuki Kawamura a , Toshio Ishikawa a , and Norito Kawakami b a National Center of Neurology and Psychiatry, Japan; b Tokyo University, JapanChronic stressors and depressive symptoms are associated with suppression of both cellular and humoral immunity. Recently low NK cell activity was shown to be associated with increased cancer risk. In addition, it is reported that critical life events and depressive symptoms are the risk factors for cancer incidence. Thus, one can hypothesize that stressors and/or stress symptoms are one of the major risk factors via stress-induced immuno-suppression.In 1997, we have started an epidemiological cohort study in order to elucidate the relationship between stress, immunity and cancer. Following approval of the ethical aspects of this study by the institutional review boards, we administered questionnaires to 4392 workers, aged 20 to 60 years old, working at a company manufacturing motor vehicle parts and collected blood samples from the 3790 (86.3%) responders who gave written informed consent. Information on the questionnaires included gender, birth date, lifestyle habits, marital status, education., family history of cancer, past and current history of diseases and job stress. At baseline, we measured lymphocyte subpopulations for all the participants, whereas cytokine and NK cytotoxity assays for 247 of participants randomly selected from the total subjects.According to the follow-up data, we have found that depressive symptoms were associated with increased cancer incidence, lower NK cell activities and lower cytokine productions, indicating that the relevance of hypothesis. CS14-01 Loss of axonal Na + /K + ATPase in chronic lesions of multiple sclerosis Christine Fowler, Elizabeth A.Young, Graham Kidd, Ansi Chang, Bruce D. Trapp Department of Neurosciences, Cleveland Clinic, Cleveland, OH, USA Neurodegeneration is the major cause of permanent neurological disability in individuals with the demyelinating disease, multiple sclerosis. The continuous and irreversible neurological decline that occurs during the latter stages of MS is thought to result from degeneration of chronically demyelinated axons. The molecular mechanisms responsible for the degeneration or dysfunction of chronically demyelinated axons are poorly understood. One proposed mechanism involves dysfunction of the Na + /K + ATPase, which is essential for nerve transmission. In this study, we determined the distribution of Na + /K + ATPase subunits in normal human white matter and demyelinated lesions in multiple sclerosis. Na + /K + ATPase subunits α1, α3 and β1 were detected in the internodal axolemma and absent from the nodal axolemma of myelinated fibers in the adult human brain. Following demyelination, all three subunits were initially detectable on demyelinated axolemma. In contract, over 50% of demyelinated axons in chronic inactive lesions of multiple sclerosis contained little or no Na + /K + ATPase immunoreactivity. As a result, these chronically demyelinated axons cannot exchange axoplasmic Na + for extracellular K + and their axolemma remains in a depolarized state and incapable of nerve transmission. We propose that loss of axonal Na + /K + ATPase is a major contributor to axonal degeneration and continuous neurological decline in multiple sclerosis patients. CS14-02 Oligodendrocytes and Schwann cells support axon function and survival independent of their role in myelination: Implications for multiple sclerosis Oligodendrocytes and Schwann cells are well known for their common function in faciliting the rapid impulse propagation in the nervous system, by enwrapping axons with myelin. Experimental genetic data suggest there are additional functions of ensheathing glia that may even precede myelination (in evolutionary terms) and are relevant for disease. We have previously shown that null mutations of two glial genes, Plp1 and Cnp1, do not impair the functional myelination of the CNS but cause progressive axonal degeneration in the absence of inflammation (Griffiths et al., Science, 1998; Lappe-Siefke et al., Nat Genet., 2003) . Axon loss is preceded by swellings and slowing of retrograde transport (Edgar et al., JCB, 2004) , but the underlying cause is not understood. This suggests that some oligodendrocyte functions are coupled to the energy state of the white matter. In support, we find extensive axonal loss and demyelination in Pex5 conditional mouse mutants that lack functional peroxisomes, which are known for their role in fatty acid β-oxydation. Surprisingly, the oligodendrocyte-specific loss of Pex5 shares many pathological features with human X-linked ALD, including the invasion of B-and T-cells into the brain (Kassmann et al., unpublished) . This novel mouse mutant proves experimentally that glial peroxisomes are important for axonal maintenance and that a primary oligodendrocyte defect is sufficient to cause a secondary inflammatory response, a finding relevant to the etiology of multiple sclerosis. CS14-03 Mechanisms to promote axonal regeneration in vivo Marie T. Filbin Biology Department, Hunter College, 695 Park Avenue, New York, NY 10024, USA One of the major impediments to axonal regeneration after injury is inhibitors in myelin. Three myelin inhibitors have been identified in NogoA, MAG and OMgp. One approach to overcome these inhibitors to encourage regeneration is to change the intrinsic state of the axon such that it no longer recognizes these molecules as inhibitory. We have shown that if the neuronal cAMP levels are elevated either artificially with an analogue such as db cAMP or by priming neurons with a variety of neurotrophins (NGF, BDNF) before exposure to the inhibitor, MAG and myelin in general no longer inhibit axonal growth. Furthermore, we have also shown that the longrecognized ability of spinal dorsal column axons to regenerate if the peripheral branch of the same neuron is lesioned beforehand is a consequence of a transient increase in endogenous cAMP levels in the DRG cell bodies. Importantly, injection of db cAMP directly into the DRG in the absence of a conditioning lesion, is sufficient to induce regeneration of subsequently lesioned dorsal column axons. This cAMP effect is transcription dependent and we have identified 4 very different genes that are up-regulated in response to either elevation of cAMP or a conditioning lesion. All 4 of these proteins block inhibition by myelin and they are currently being tested for their ability to promote regeneration in vivo. Franklin Cambridge Centre for Brain Repair, University of Cambridge, Cambridge CB3 OES, UK Remyelination, the process by which new myelin sheaths are restored to demyelinated axons, represents one of the most compelling examples of adult multipotent progenitor cells contributing to regeneration of the injured CNS. This process can occur with remarkable efficiency in both clinical disease, such as multiple sclerosis, and in experimental models, revealing an impressive ability of the adult CNS to repair itself. However, the inconsistency of remyelination in multiple sclerosis, and the loss of axonal integrity that results from its failure, makes enhancement of remyelination an important therapeutic objective. Identifying potential targets will depend on a detailed understanding of the cellular and molecular mechanisms of remyelination. This talk will review (1) the nature of the cell or cells that respond to demyelination and generate new oligodendrocytes, identifying current areas of uncertainty and addressing the role of adult CNS stem and progenitor cells, (2) intrinsic factors regulating precursor differentiation and (4) how an environment favourable to remyelination is generated, and will introduce the concept of a matrix of signalling events critical for the successful completion of remyelination. WS01-K1 Genetics of neuroimmunological disorders T. Olsson a and George Ebers b a Neuroimmunology Unit, Karolinska Hospital, Stockholm, Sweden; b University of Oxford, UK There is solid evidence for the role of genes regulating MS. Their deciphering may allow definition of mechanisms central for disease and thereby for more precise and selective therapy. Studies in experimental autoimmune encephalomyelitis (EAE) have shown allele specific disease protective and promoting actions from both the class I and class II genes, possibly to be conveyed by critical peptide-MHC molecule interactions. Linkage analyses have failed to show any non-HLA region and the paradigm generally used to justify genome searches may be incorrect or the effect of any individual locus would be small. Indeed, the few examples of discrete non-MHC genes with suggested or proven influence display odds ratios in the order of 1.5, such as the PKRCA, IL7R and MHCIITA. There is some interest in whole genome SNP typing with association analysis of large case-control materials but there is no assurance that this approach will be fruitful. Gene mappings in EAE may reveal genes and/or mechanisms which are shared between species with options for experimental studies of function. Collectively, there are now around 50 quatitative traits loci (QTLs) described in different rodent crosses. Upon fine dissection each of these often resolves into two to five subQTLs, showing that linkage peaks mostly depend on more than one gene and by extrapolation that the number of genes which potentially has a role in MS approaches hundreds. The experimental QTLs can be fine dissected. Albeit cumbersome, definite disease relevant variants of genes result and the first few positioned genes start to appear in the literature. In Western countries, frequencies of cases of opticospinal (OS) multiple sclerosis (MS), and of MS cases lacking oligoclonal immunoglobulin G bands (OCB) in the cerebrospinal fluid, are very low. (OSMS was defined broadly as clinical evidence of involvement of the optic nerve, brainstem or spinal cord, without clinical or radiological evidence of disease in the cerebrum or cerebellum.) Carriage rates of HLA class II alleles in these "fringe phenotypes" were compared with rates in clinically and paraclinically more typical MS. HLA genotyping was performed in subsets of patients as well as in healthy controls. Carriage of DRB1 ⁎ 04, but not of DRB1 ⁎ 15, was associated with OCB-negative MS (odds ratio [OR], 2.1; 95% confidence interval [CI], 1.2 to 3.8). High-resolution genotyping of HLA-DRB1⁎04 uncovered a highly significant riskconferring association between the DRB1 ⁎ 0404 allele and OCB-negative MS (OR, 4.3; 95% CI, 1.9 to 9.7). The frequency of DRB1 ⁎ 15 was slightly (yet non-significantly) higher in OSMS than in conventional MS. In conclusion, in Sweden, the fringe phenotype OSMS is not uniquely associated with any HLA-DRB1 allele; OCB-negative MS, however, appears to be immunogenetically distinct from OCB-positive MS. Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS. Complex diseases such as MS likely result from problems in networks of interactions between several genes and environmental factors. HLA-DRB1 ⁎ 15 is the only consistent locus observed in most populations, but numerous genome screens also provide evidence for a MS locus on 17q.Sixty-three Finnish MS families were ascertained to identify a susceptibility gene within the previously established 3.4-Mb region on 17q24, which is flanked by segmental duplications. Such a complex genome structure may predispose to copy-number or other structural variation. Initial association implicated PRKCA, which was further analyzed in 211 Finnish and 554 Canadian MS families, utilizing a dense SNP set. SNP haplotype analysis revealed two allelic variants of PRKCA to be over-represented in Finnish or Canadian MS cases (odds ratios: 1.34 and 1.64 ). An analysis of 200 MS families from Sweden, Denmark and Norway suggested a role for the Finnish risk haplotype in MS susceptibility also in other Scandinavian populations. Transcript levels of PRKCA correlated with the copy number of the risk alleles in 20 Finnish and 11 CEPH individuals of European origin in an initial expression analysis.These results imply involvement of PRKCA in the etiology of MS. Moreover, function of PRKCA in a signal transduction pathway altering blood-brain barrier permeability supports its role in chronic inflammation of CNS and myelin destruction. WS01-03 Protective influence on multiple sclerosis by HLA-A ⁎ 02: Implications for a two step model of pathogenesis Multiple sclerosis (MS) was traditionally thought to be a CD4 + T-cells mediated disease, associated with HLA-DRB1 ⁎ 15. However, a two-step model, recently proposed by Friese (2005) , implicates CD4 + T cells in the beginning of MS and CD8 + T-cells in the chronic phase of the disease. This model suggests that HLA-class I may also contribute to MS, as already described for HLA-A ⁎ 03 and HLA-A ⁎ 02.The aim of this study was to investigate the role of HLA-A ⁎ 02 and HLA-A ⁎ 03 alleles on disease susceptibility and severity in a Portuguese MS population.We studied 242 unrelated patients with clinically definitive MS according McDonald criteria, attending the outpatient neurological clinic of HGSA and 142 healthy controls from North of Portugal. To evaluate disease severity, patients were divided in 3 groups: 44 have Benign MS (EDSS ≤ 3, ≥ 10 years after onset), 36 Non-Benign MS (EDSS ≥ 3, at same stage) and 41 Aggressive MS (EDSS ≥ 6, ≤15 years after onset).HLA-A ⁎ 03 frequency was similar in patients and controls. This difference was even higher in DR15 negative individuals (32.9% vs. 57.0%, OR = 0.370, p = 0.00006). When disease severity was considered this association was observed only in Benign group (34.1% vs. 52.7%, OR = 0.464, p = 0.030).Our results are in accordance with Fogdell-Hahn (2000) establishing HLA-A ⁎ 02 allele as a protective genetic marker to MS in this Portuguese population. This observation supports the Friese model for MS, highlighting the importance of HLA-Class I in CD8 T-cells function and its role in disease outcome.Work supported by Serono Portugal. WS01-04 Idd5.4 on chromosome 1 mediates resistance to experimental autoimmune encephalomyelitis Idd5, a type 1 diabetes (T1D) susceptibility region, is located on chromosome 1. Introgression of DNA from T1D-resistant strain onto the NOD background protects from not only T1D but also experimental autoimmune encephalomyelitis (EAE). To study the Idd5 region for disease susceptibility, congenic mice having the entire locus and subcongenic lines having defined portions of the region were generated. Our previous study showed that Idd5.1, which is the most proximal Idd5 and contains the Ctla4 and Icos, enhanced EAE severity and regulated ICOS expression and IL-10 production. As compared to NOD-derived T cells, T cells from the EAE-resistant congenic mice produce lower amounts of Th1 and higher amounts of Th2 cytokines. This cytokine alteration may be responsible for disease protection in the EAE-resistant congenic mice. Genetic elements present in the region for the disease resistance have not been characterized. The identity of the gene causing EAE resistance will require the development of additional congenic strains to reduce the size of the subregion and thereby the number of candidate genes, one of which encodes the T cell inhibitory molecule PD-1. Whether a polymorphism in PD-1 impart genetic resistance and affects Th1/Th2 differentiation is being studied. WS01-05 Evidence the Y-chromosome influences experimental allergic encephalomyelitis in female mice Experimental allergic encephalomyelitis (EAE), an autoimmune model of multiple sclerosis, is a complex disease influenced by genetic, intrinsic and environmental factors. In this study, we questioned whether parent-oforigin effects influence EAE, using reciprocal F2 intercross progeny generated between EAE susceptible SJL/J (S) and EAE resistant B10.S/ SgMcdJ (B) mice. EAE susceptibility and severity was found to be different in female BS × BS intercross mice as compared to females from the three other birth crosses (BS × SB, SB × SB and SB × BS), and in fact, both traits in female mice resembled those of their male siblings. Related studies using C57BL/6J Ychromosome substitution strains demonstrate that the Y-chromosome again influences EAE in both male and female mice, and that the disease course and MOG 35-55 specific immune response in females resembles that of their male littermates. Importantly, this is the first experimental evidence demonstrating the existence of a Y-chromosome polymorphism capable of modifying autoimmune disease susceptibility in both males and females. During development of the CNS the chemokine receptor CXCR2 controls positioning of oligodendrocyte precursors by arresting their migration. CXCR4 is essential for forming the normal cerebellar cytoarchitecture by regulating proliferation and migration cerebellar granule cells.In the healthy adult brain certain chemokines like CXCL12 (on astrocytes, endothelial cells) and CX3CL1 (on neurons) are displayed. Neuronal derived CX3CL1 can be shed and regulates the state of activation of microglia.A production of chemokines inside of the CNS by astrocytes and microglia is induced in autoimmune inflammation, infection and also after neurotrauma. Chemokine production is stimulated via T cell derived cytokines and via innate immunity. The role of chemokines in CNS infections is indicated by the observation that human subjects with a loss-offunction mutation of CCR5 have a higher risk of developing encephalitis after infection with West Nile virus. In EAE models and in MS the inflammatory cytokines CXCL10/IP10, CCL2/MCP-1, CCL5/RANTES, but also the homeostatic chemokines CXCL12/SDF-1, CXCL13/BCA-1, and CCL19/Mip3b are involved in lesion formation.The crucial role of chemokines in neuroinflammation provides the possibility to develop novel chemokine-directed therapeutic strategies. WS02-01 The chemokine CXCL13 is a key molecule in autoimmune Myasthenia Gravis A. Meraouna 1 , G. Cizeron-Clairac 1 , R. Le Panse 1 , J. Bismuth 1 , F. Truffault 1 , C. Tallaksen 2 and S. Berrih-Aknin 1 1 CNRS-UMR 8162, IPSC, Le Plessis-Robinson, France (sonia. berrih@ccml.u-psud.fr); 2 Department of Neurology, Ulleval University Hospital, N-0407 Oslo, Norway Myasthenia Gravis (MG) is associated with ectopic germinal centres in the thymus. Thymectomy and glucocorticoids are the main treatments but they induce operative risks and side effects, respectively. The aim of this study was to propose new therapies more efficient for MG. We hypothesized that molecules dysregulated in MG thymus and normalized by glucocorticoids may play a key role in thymic pathogenesis. Using gene chip analysis, we identified 88 genes complying with these criteria, the most remarkable being the B cell chemoattractant (CXCL13). Its expression was increased in thymus and sera of glucocorticoid untreated patients and decreased in response to treatment in correlation with clinical improvement. Normal B cells were actively chemoattracted by thymic extracts from glucocorticoid untreated patients, an effect inhibited by anti-CXCL13 antibodies. In the thymus, CXCL13 was preferentially produced by epithelial cells and overproduced by epithelial cells from MG patients. Altogether, our results suggest that a high CXCL13 production by epithelial cells could be responsible for germinal centre formation in MG thymus. Furthermore, they show that this gene is a main target of corticotherapy. Thus, new therapies targeting CXCL13 could be of interest for MG and other autoimmune diseases characterized by ectopic germinal centre formation. The role of CXCR3 ligands, CXCL9 and CXCL10, in herpes simplex virus type 1 (HSV-1) infection was investigated. C57BL/6 wild type (WT), CXCL9 deficient (CXCL9 −/− ), and CXCL10 deficient (CXCL10 −/− ) mice were infected with HSV-1 (1000 plaque forming units/cornea) and assessed for virus titer as well as the host response to infection in the trigeminal ganglion (TG) and brain stem (BS) during acute infection (days 3-7 post infection, pi). HSV-1 titers recovered in the TG and BS of CXCL10 −/− mice were 1-2 logs higher in comparison to CXCL9 −/− or WT mice day 3-day 7 pi. Likewise, there was a significant loss in thymocytes in the CXCL10 −/− mice following infection relative to the other two groups which did not correlate with changes in corticosterone levels but rather, the incidence of detectable virus within the thymus. Bone marrow progenitor cell commitment was noticeably modified with a strong change toward myeloid and away from lymphoid progenitors. In evaluating T cell infiltration into the TG and BS of infected mice, CXCL9 −/− mice possessed more CD4 and CD8 T cells in the TG compared to the WT mice day 7 pi. In contrast, CXCL10 −/− mice possessed more CD4 and CD8 T cells in the BS compared to the WT mice day 7 pi. The absolute number of NK cells (NK1.1 + CD3 − ) was reduced in the TG and BS of CXCL10 −/− mice compared to the WT or CXCL9 −/− mice. WS02-03 Differential effects of decoy chemokine (7ND) gene therapy on acute, biphasic and chronic autoimmune encephalomyelitis: Implication for the pathomechanisms of lesion formation Il-Kwon Park, Kuniko Kohyama, Mie Nakajima and Yoh Matsumoto Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, JapanMultiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), exhibit several clinical subtypes such as relapsing-remitting and secondary progressive forms. Although clinical signs are similar among disease subtypes, it becomes evident that the lesion formation in the central nervous system (CNS) may take place by different mechanisms. In the present study, we induced three different types of EAE, i.e. acute, biphasic and chronic EAE, in rats and examined the effects of decoy chemokine (7ND) gene therapy, which inhibit the migration of macrophages. Interestingly, it was demonstrated that the clinical course of acute EAE was not affected, whereas chronic EAE was completely suppressed with the treatment. Furthermore, the relapse, but not the first attack, of biphasic EAE was inhibited by gene administration. Histopathological examinations revealed that the number of infiltrating macrophages was reduced in all the stages of three types of EAE. Real time PCR analysis showed that the levels of cytokine and chemokine mRNA paralleled with the macrophage behavior in the CNS. These findings suggest that in acute EAE and the first attack of biphasic EAE where anti-macrophage therapy was almost ineffective, pathogenic T cells play a critical role. In contrast, the relapse of biphasic EAE and chronic EAE were thought to be produced mainly by macrophages. Thus, the mechanisms of the lesion formation are not uniform and immunotherapy should be performed on the basis of information about the pathomechanisms of the lesion formation in autoimmune diseases. WS02-04 SDF-1α modulates the innate rhythm of network-driven GABA-mediated giant depolarizing potential in the developing nervous system A. Kasiyanov 1 , H.Tamamura 2 , N. Fujii 3 , H. Xiong 1 1 University of Nebraska Medical Center, Omaha, USA; 2 Tokyo Medical and Dental University, Tokyo, Japan; 3 Kyoto University, Kyoto, JapanThe chemoattractant stromal cell-derived factor-1 alpha (SDF-1α) and its receptor, the CXC chemokine receptor 4 (CXCR4), play an important role in the formation of hippocampal circuitry and synaptic plasticity in addition to their involvement in brain inflammatory processes. To understand the role that CXCR4 might play in hippocampal circuitry development, we studied the effects of SDF-1α, the only natural ligand for CXCR4 receptors, on network-driven, GABA-mediated giant depolarizing potentials (GDPs) in immature rat hippocampal CA3 neurons using whole-cell patch clamp technique. We demonstrated that SDF-1α down-regulates the GDP firing frequency in CA3 neurons without significant change of neuronal passive membrane properties, suggesting that the site of action for SDF-1α is on interneurons and/or glial cells, instead of the recorded neuron per se. The SDF-1α-induced down-regulation of GDP firing was blocked by T140, a CXCR4 receptor antagonist. Taken together, our results showed that exogenous and endogenous SDF-1α modulates GDP firing via CXCR4 receptors in developing hippocampus. WS02-05 The role of CX3CR1 in aminoglycoside ototoxicity Our recent studies demonstrated the accumulation of CD45 + , CD68 + , CX3CR1 + and Iba-1 + macrophages in the cochlea, following acoustic trauma. It is unknown whether inflammatory cells play a role in ototoxic injury. This study was designed to investigate what role CX3CR1 plays in hearing loss and hair cell death after aminoglycoside exposure. CX3CR1 +/+ , CX3CR1 +/− and CX3CR1 −/− mice at age 8 weeks were exposed to 900 mg/kg kanamycin intraperitoneally (IP) delivered twice daily for 15 days. Hearing thresholds were recorded 14 days after the final dose of kanamycin (age 12 weeks), and cochleas were harvested for histologic analysis. Hearing thresholds in CX3CR1 −/− mice were profoundly elevated when compared to either the wild type or the CX3CR1 +/− mice. In addition, hair cell damage was greatest in the CX3CR1 −/− mice when compared to their wild type and heterozygous littermates. Neither hair cell numbers nor hearing thresholds were abnormal in control CX3CR1 −/− mice which were not exposed to aminoglycoside. Our data indicate a protective role for CX3CR1 in aminoglycoside ototoxicity, possibly acting through cochlear macrophages, to reduce hair cell death and lessen hearing impairment.Over the last 20 years, there has been a dramatic re-evaluation of the type of cellular immune responses that can and do occur within the central nervous system (CNS). For the better part of the last century, the CNS was believed to be both immunologically inert and immunologically separated from the peripheral immune system. It was viewed primarily as a tissue of post-mitotic cells that were highly vulnerable to the onslaught of activated immune cells if and when these immune cells infiltrated the CNS. In this view, the only elements protecting the CNS from immune-mediated harm was the presence of an intact blood-brain barrier (BBB) and the absence of a fully immunologically population of competent tissue macrophages. Current data now indicates that the CNS is immunologically competent and is actively interactive with the peripheral immune system. Inflammation is also now recognized to be a prominent feature of many classic neurodegenerative disorders even in the absence of substantial infiltration of peripheral immune cells. Furthermore, in stark contrast to the older view, neuroinflammation is now realized to have both neuroprotective as well as neurotoxic aspects. Here we will discuss new classes of receptors (TREMs, TLTs and LR8) with the potential to trigger and modify glial inflammatory responses. WS03-01 Identification of soluble CD14 as an endogenous agonist for Toll-like receptor 2 on human astrocytes by genome-scale functional screening of glial cell derived proteins Human astrocytes express a very limited repertoire of Toll-like receptor (TLR) family members including TLR1-4, which are expressed on the cell surface. Recent data indicated that TLRs on astrocytes not only play a role in host defense but also in tissue repair responses. This prompted us to examine the possibility that endogenous TLR agonists could be expressed in the human central nervous system to regulate the apparently dual astrocyte functions during trauma or inflammation.We developed a high throughput, genome-scale functional screening method to identify these endogenous TLR agonists. The method is based on the pooled transfection of brain-derived genes in eukaryotic cells and the evaluation of functional effects of translated proteins on primary human astrocytes. IL-8 release was used as read-out, since this cytokine is highly produced by astrocytes upon activation via TLR. Using this strategy, we identified an alternatively spliced variant of soluble CD14. Using a panel of TLR transfected HEK293 cells, we found that soluble CD14 only induced IL-8 release by TLR2 transfected HEK293 cells, and not TLR3 or TLR4 transfected HEK293 cells.These data indicate that apart from its well-known ability to act as a coreceptor for TLR-dependent signaling by peptidoglycans or LPS, soluble CD14 itself also acts as a direct agonist for TLR2. Currently, we are further exploring the functional effects of soluble CD14 on human primary astrocytes. The genome-scale screening method will be used to identify proteins based on their functional properties. We previously reported that Toll-like receptor (TLR) ligation tailors the activation profile of human microglia; TLR3, in particular, induces the production of both IL-12 and IFN-beta (â). The functional consequences to adaptive immune responses downstream of such TLR-mediated polarization remain undefined in human cells. We compared the capacity for adult human microglia, once activated in vitro with ligands for TLR2, TLR3, or TLR4, to regulate CD4 + T cell responses, specifically Th1-priming. We tested the ability for these ligands (poly(inosinic):poly(cytidylic) acid for TLR3, lipopolysaccharide for TLR4, palmitoyl-3-cysteine-serine-lysine-4 for TLR2) to induce the maturation of microglia in terms of MHC and costimulatory molecule expression using flow cytometry. The proliferation and interferon-gamma (IFNã) secretion of allogeneic CD4 + lymphocytes was determined following culture with microglia pre-activated via TLR2, TLR3 or TLR4 for 24 h. We found that TLR signaling increased microglial MHC class I and II, CD80 and CD86 surface expression downstream of TLR3 and TLR4. While TLR-mediated activation of adult microglia did not result in any significant increase in overall CD4 + T cell proliferation, the ligation of TLR3 by microglia led to a significant boosting of IFNã secretion by T cells. TLR-signaling can thus lead to the maturation of human microglia as antigen presenting cells, and TLR3mediated activation can specifically bridge innate and adaptive immune responses by driving Th1 T cell polarization. WS03-03 A critical role of toll-like receptor 2 in nerve injury-induced spinal cord glial cell activation and pain hypersensitivity The activation of spinal cord glial cells has been implicated in the development of neuropathic pain upon peripheral nerve injury. The molecular entity that activates glial cells, however, has not been elucidated. To address this lack of information, primary spinal cord glial cells were stimulated with various molecules that are putatively released upon peripheral nerve injury in the spinal cord dorsal horn. Our in vitro data indicate that spinal cord glial cells are relatively unresponsive to the stimulation of glutamate, substance P, ATP, lysophosphatidic acid (LPA), and fractalkine. Interestingly, necrotic neuronal cell extract (NNCE), however, induced significant upregulation of the TNF-α, IL-1β, IL-6, and iNOS genes that are implicated in the development of neuropathic pain. Studies using primary glial cells isolated from TLR2 knockout mice indicate that NNCE activate glial cells via TLR2. In addition, behavioral studies using TLR2 knockout mice demonstrate that the expression of TLR2 is required for the induction of mechanical allodynia and thermal hyperalgesia due to spinal nerve axotomy. The nerve injury-induced spinal cord microglia and astrocyte activation is reduced in the TLR2 knockout mice. Similarly, the nerve injury-induced proinflammatory gene expression in the spinal cord is also reduced in the TLR2 knockout mice. These data demonstrate that TLR2 plays a critical role in nerve injury-induced spinal cord glial cell activation and subsequent pain hypersensitivity. In addition, our data imply that endogenous TLR2 agonist, released from the damaged sensory neurons, may activate spinal cord glial cells via TLR2 and thereby induce neuropathic pain. WS03-04 Microglial activation is correlated with decreased expression of EAAT-2 in the cerebral cortex of HIV-1 infected patients: A neuroprotective role of microglia in AIDS encephalopathy Izumo S a , Xing HQ a , Kuboda R a , Hayakawa H a,b , Gelpi E b , Budka H b a Center for Chronic Viral Diseases, Kagoshima University, Kagoshima, Japan; b Institute of Neurology, Vienna University, Vienna, Austria Pathogenesis of AIDS encephalopathy has been discussed in correlation with microglial activation and infiltration of HIV-1-infected macrophages. Glutamate transporter-1 (EAAT-2) was primarily expressed on astrocytes and keeps extracellular glutamate concentration low in the brain by taking up glutamate, which prevent neurons from excitotoxic cell death. Gray and Gras reported that both microglia and brain macrophages expressed EAAT-2 and have a neuroprotective role in AIDS encephalopathy. In order to clarify precise roles of microglia in AIDS encephalopathy, we examined autopsy brains of twenty patients with HIV-1 infection by immunohistochemistry. Expression of IL-1β and TNF-α were detected only in glial nodules, but not in diffusely activated microglia. These cortical changes were neither correlated with severity of HIV encephalitis nor presence of HIV-1-infected cells. A quantitative analysis of EAAT-2 expression and microglial activation demonstrated that the number of Iba1-positive activated microglia was increased in 12 cases and the area of EAAT-2 expression was declining in 12 cases. There was a significant negative correlation between areas of EAAT-2 expression and numbers of Iba1-positive activated microglia (P < 0.01) among the cases with decreased EAAT-2 expression. These data indicate that activation of microglia occurs according with reduction of EAAT-2 expression on astrocytes in the cerebral cortex of AIDS patients. WS03-05 TrkB is physiologically expressed on astrocytes and is upregulated in multiple sclerosis lesions Neurotrophins play a fundamental role in regulating proliferation and differentiation of neurons. They exert their actions through the p75 receptor or the different Trk receptors. Little is known about their potential effects on glia cells. Published data indicate that astrocytes express neurotrophin receptors (NTR) mainly after activation or neoplastic transformation.The aim of the present study was to analyse the NTR profile in human astrocytes in vitro and in situ in normal and in multiple sclerosis (MS) brain samples.Molecular analyses showed that resting foetal astrocytes transcribed mRNA for p75, TrkB and TrkC, but not TrkA. When TrkB and TrkC isoforms were analysed by real time PCR, mainly the truncated isoforms were detectable in resting astrocytes. Exposure of the cells to IFN-gamma enhanced p75 levels at the mRNA and at the protein levels, whereas TrkB and TrkC were unchanged. Immunofluorescence showed membrane staining for all three receptors. Furthermore immunohistochemical analysis showed constitutive expression of TrkB in situ on astrocytes in normal brain specimens and enhanced levels within MS lesions.Together, these data suggest a role of neurotrophins in regulation of glia cell reactivity.Workshop 4: Gene therapy and immunotherapy for brain tumors 47 Abstracts WS04-K1 Gene therapy and immunotherapy for brain tumors Kyogo Itoh a,b and Joseph C. Glorioso b a Immunology, Kurume University School of Medicine, Japan; b Molecular Genetics and Biochemistry, University of Pittsburgh Medical Center, USA Development of new treatment modality is needed to improve the prognosis of brain tumors. Recent progress of basic and clinical research in the field of neuron oncology suggests that both gene therapy and immunotherapy could have promising features as new treatment modalities. This workshop takes five distinguished presentations related on gene therapy and immunotherapy for brain tumors. The first presentation reports new findings on glioma-infiltrating dendritic cells, while the second one shows that the invasion promoting effect of microglia on glioblastoma cells is suppressed by cyclosporine A. The third paper presents findings supporting a paradigm in which galectin-9 expression on low grade gliomas inactivates tumor-infiltrating T cells, which in turn results in promotion of tumor cell proliferation and invasiveness, using a CD4 + T cell clone established from glioblastoma tissue. The last two papers reports new data related to gene therapy. The fourth paper shows new findings on HSV vector-mediated co-delivery of mutant IkBa and HSV thymidine kinase against glioblastoma, while the last presentation indicates that suicide gene therapy using high-titer retrovirus vector is cytotoxic to the cancer stem cells derived from malignant gliomas. We hope active discussion on each of the five distinguished presentations to better understand their potential for development of clinically effective treatment modalities to glioblastoma multiforme. WS04-K2 HSV gene vectors and treatment of chronic pain and cancer Herpes simplex virus is a promising vehicle for delivery of genes to the nervous system. Our laboratory has exploited replication defective vectors for treatment of peripheral nerve diseases that include chronic pain and peripheral neuropathy and for treatment of central nervous system diseases that include cancer and Parkinson's disease. Experiments will be presented that describe current vector engineering for these applications and specific studies related to the treatment of chronic pain using enkephalin gene vectors. In addition, given the interest in the use of oncolytic HSV vectors for treatment of cancer, I will describe new vectors that rely on mutations affecting ICP0 and ICP22 for virus attenuation. These vectors have a highly improved profile for both safety and efficacy in brain tumor therapies. Objective: Although many mechanisms are responsible for aggressive behavior of glioblastomas, the role of systemic immunsuppression by this tumors appears to be a critical component of the pathogenesis. Dendritic cells (DCs) play a critical role in orchestrating anti-tumor immunity.Previous work implicates defects and/or impaired maturation/migration of DCs as a critical defect in the immune response against cancer. The aim of this study was to identify and characterize glioma infiltrating DCs by immunohistochemistry (IHC), FACS and functional assays. Methods: IHC was performed on 24 gliomas using DC-specific antibodies and the infiltrating mononuclear cell populations derived from six tumors were evaluated by three-color-FACS analysis. In addition, the stimulatory capacity of monocyte-derived DCs from tumor patients were compared with controls in an allogenic MLR and the levels of IL-12 and IFN-α production were assessed by ELISA. Results: DCs were found to be present in high grade gliomas at very low numbers and mainly in an immature state. The origin of glioma-derived DCs was mainly myeloid and the T cell stimulatory capacity was strongly reduced in allogenic MLR. Interestingly, the capacity of monocyte-derived DCs of patients was also reduced albeit not being statistically significant to controls. Conclusion: Taken together, these results suggest that the inhibition of DC development/function represents an additional mechanism by which gliomas can escape immune recognition. WS04-02 The invasion promoting effect of microglia on glioblastoma cells is inhibited by cyclosporine A Tumor cells seem to recruit stromal and inflammatory cells to the tumor site and transform into tumor-supportive cells. Invasion of glioma cells into brain tissue is a hallmark of glioblastomas and contributes to the failure of current therapeutic treatments. However, defense functions of microglial cells against glioma are compromised by the tumor, e.g. through impaired surface expression of MHC class II, the immune-suppressed microglia may still display tumorpromoting activity. We found that Cyclosporine A (CsA) can affect growth of glioma cells in vitro by inhibiting signaling pathways, which are essential for tumor proliferation and invasiveness. In this work, we demonstrate that migration of EGFP-transfected glioblastoma cells in organotypic brain slices was significantly inhibited by 1-30 μM CsA. This inhibitory effect was lost when glioblastoma cells were injected into microglia-depleted brain slices. In in vitro studies we demonstrate that microglia-derived factors increase glioma invasiveness in Matrigel assays which is associated with activation the PI-3K/Akt signaling pathway. On the other hand, glioma-derived soluble factors induce morphological transformation of microglia into ameboid microglia, activate MAPK signaling, although production of pro-inflammatory factors such as nitric oxide or IL-6 was not observed. We found that CsA may interfere with glioma-associated microglia stimulation, turning them into more resting state. Our finding that CsA may impair invasive growth of glioma cells, at clinically relevant concentrations, provides a novel therapeutic strategy. Gal-9 expression by peripheral human tumors has a favorable clinical prognosis and appears to limit tumor cell metastasis/invasiveness. Thus, one prediction is that glioma expression of Gal-9 limits tumor cell proliferation and/or invasiveness. Indeed, we have found that siRNA-mediated reduction of Gal-9 in a primary GBM cell line enhances tumor cell proliferation. Moreover, culture of this cell line in TGF-β, which is expressed at higher levels in high-grade versus low-grade gliomas, reduces tumor cell expression of Gal-9. In addition, quantitative RT-PCR analysis of Gal-9 levels expressed by GBM tumor cells versus reactive astrocytes indicates that GBM tumor cells express lowers levels of Gal-9 in situ. On the other hand, Gal-9 has recently been identified as a ligand of TIM-3, and been shown to kill IFN-γ-secreting Th1 cells. To this end, we have cloned CD4 + T cells that infiltrate GBMs and found that exposure to recombinant Gal-9 inhibits IFN-γ secretion from activated Th1 cells, which can be blocked with anti-TIM3 monoclonal antibody. Collectively, the data suggest a paradigm in which Gal-9 expression by low-grade gliomas inactivates infiltrating TIM3 + CD4 + and CD8 + T cells, and that with increasing stages of malignancy, down-modulation of Gal-9 on gliomas promotes tumor cell proliferation and invasiveness. WS04-04 Combination gene therapy for glioblastoma involving herpes simplex virus (HSV) vector-mediated co-delivery of mutant IκBα and HSV thymidine kinase against glioblastoma To improve the effectiveness of HSV thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene therapy, the replication defective HSV vector TOIκB expressing both HSV-TK and a mutant form of the NF-κB inhibitor IκBα (IκBαM) was developed. In human glioblastoma U-87MG cells, the p50/p50 dimer of NF-κB was already translocated to the nucleus without receptor-dependent signaling by TNF-α. Following infection with TOIκB, nuclear translocation of NF-κB in U-87 MG cells was significantly inhibited and caspase-3 activity increased compared with TOZ.1-infected cells. The cytotoxicity of TOIκB for U-87 MG cells was investigated by colorimetric MTT assay. At an MOI of 3, TOIκB infection killed 85% of the cells compared to 20% killed by TOZ.1 infection. In the presence of GCV, these numbers increased to 95-100% for TOIκB and 80-85% for TOZ.1. The survival of nude mice implanted into the brain with U-87MG tumor cells was markedly prolonged by intratumoral TOIκB injection and GCV administration. Survival of TOIκB + GCV group was significantly longer (P < 0.02, Wilcoxon test) than for the control groups (TOZ.1 or TOIκB only, PBS or PBS + GCV). These results suggest that IκBαM expression may be a safe enhancement of replication-defective HSV-based suicide gene therapy in vitro and in vivo. WS04-05 Efficacy of suicide gene therapy using high-titer retrovirus vector for cancer stem cells derived from malignant glioma T. Yawata a , K. C. Park a , S. Toyonaga a , T. Chihara a , N. Masahira a , K. Ikenaka b , K. Shimizu a a Department of Neurosugery, Kochi Medical School, Nankoku, Japan; b Division of Neurobiology and Bioinformatics, National Institute for Physiological Science, Okazaki, Japan Despite many efforts to develop effective therapy, the outcome of malignant glioma remains poor. Gene therapy for this disease using retroviral vector is attractive, because the virus can infect only mitotic cells. We established the packaging cell line PAMP51 producing high-titer retrovirus expressing suicide gene HSV-TK and concentrated the virus with titers as high as 1-10 11-12 c.f.u./ml by low speed centrifugation. Recently, several investigators identified a small population of cancer stem cells in brain tumor tissues and cell lines. These cancer stem cells forms spheres and maintains self-renewal capacity, tumorigenecity and multiple drug resistance. The existence of the cancer stem cells is thought to be a cause for recurrence of the tumors after treatment with chemotherapy, radiotherapy and surgical resection. Therefore, we investigated the therapeutic efficacy of suicide gene therapy for the cancer stem cells derived from glioma cells. A high transduction rate and gap junction mediated bystander effect allowing killing of non-transduced cells enhance the efficacy of this suicide gene therapy. A lower efficiency of transduction with retrovirus vector and the expression of connexin 43, a component of gap junction were observed in cancer stem cells. This result suggests that non-transduced cancer stem cells can be killed via bystander effect. To gain insight about bystander effect in cancer stem cells, we are currently performing immunohistochemistry studies in mouse glioma model. Studies undertaken in the speaker's laboratory have shown that a distinct population of regulatory cells can be induced by injection of soluble peptides. Such peptide treatment led to the generation of regulatory T (PI-T reg ) cells that were anergic, failed to produce IL-2 but responded to antigen by secreting IL-10. The cells were predominantly CD25 − and CTLA-4 + and their anergic state was reversed by addition of IL-2 in vitro. PI-T reg cells were able to suppress both proliferation and IL-2 production from naïve T cells in vivo, suppression being abrogated by neutralisation of IL-10. Depletion of CD25 + cells did not affect the suppressive properties of PI-T reg cells. Furthermore, PI-T reg cells could be generated in mice that do not spontaneously generate CD25 + regulatory cells. These results suggest that 'natural' and 'induced' regulatory cells fall into distinct subsets. This distinction has recently been confirmed by demonstrating that PI-T reg cells do not express FoxP3, a member of the forkhead-winged helix family of transcription factors that controls differentiation of 'natural' CD25 + T reg cells.Recent work has led to the identification of a novel set of genes upregulated in T cells rendered tolerant by peptide therapy. Some of these genes control cell division and/or cytokine production and thereby control tolerance. Clarification of the mechanisms controlling antigenspecific tolerance will lead to the development of safer and more effective vaccines for the treatment of allergic and autoimmune diseases. WS05-01 A new model of experimental autoimmune encephalomyelitis (EAE) mediated by memory CD4 cells: Differential functions of co-stimulatory pathways in regulating autoreactive memory CD4 + T cells in vivo W. Elyaman a , P. Kivisäkk a , Vijay K. Kuchroo a , Mohamed H. Sayegh b and Samia J. Khoury a a Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA; b Transplantation Center, Brigham and Women's Hospital and Children's Hospital, Boston, MA, USA A limitation of many experimental models of autoimmune disease is that they focus on targeting prevention of disease or on therapy of early disease triggered by naïve T cells. Memory T cells are less dependent on positive co-stimulatory signals than naïve cells and also less susceptible to tolerance induction. An important clinical goal is to develop strategies that suppress the function of chronically activated memory T cells. We developed a new model of EAE that is induced by antigen-specific memory CD4 + cells generated from mice immunized with MOG > 100 days prior to transfer. Memory CD4 + cells induced more severe EAE than effector T cells, both clinically and pathologically. T cells from memory cell recipients proliferated more in response to MOG and secreted higher levels of IL-2, IL-17 and IFN-γ compared to recipients of recently generated effector cells. Percentages of CD4 + CD25 + Foxp3 + cells were not affected, indicating that differences in clinical disease were not due to differential expansion of regulatory T cells. Effector cell-mediated EAE was suppressed by CTLA4-Ig, but aggravated by anti-ICOS-L treatment. By contrast, memory cellmediated disease was suppressed by ICOS-L blockade, while CTLA4-Ig had no effect. A shift in the cytokine balance towards increased IL-10 and reduced IFN-γ secretion was observed in effector cell recipients after CTLA4-Ig treatment, while ICOS-L blockade resulted in a similar shift in memory cell recipients. Our data suggest the presence of specific mechanisms of disease mediated by memory CD4 + cells and indicate a differential role of co-stimulatory pathways in regulating effector versus memory T cells. WS05-02 Differential regulation of IL-12/IFN-γ and IL-23/IL-17 axes might account for discrepancy in susceptibility towards experimental autoimmune encephalomyelitis between Albino Oxford and Dark Agouti rats We have previously shown that Albino Oxford (AO) rats are resistant to induction of experimental autoimmune encephalomyelitis (EAE). In this study we compared the production of cytokines with the presumed role in the pathogenesis of EAE, namely interferon γ (IFN-γ) and interleukins (IL) 12, 17 and 23, in AO rats and EAE susceptible Dark Agouti (DA) rats. To this end, draining (popliteal) lymph node cells (DLNC) were collected 6 days post-immunization with spinal cord homogenate and carbonyl iron as an adjuvant. The cells were stimulated with concanavalin A (ConA) or specific antigen myelin basic protein (MBP) and the production of cytokines was measured on mRNA and protein level using RT-PCR and ELISA, respectively. DLNC of DA rats expressed higher levels of mRNA and produced more IFN-γ and IL-17 than AO rats. Since IL-12 and IL-23 are potent inducers of IFN-γ and IL-17 synthesis, respectively, we further analyzed mRNA expression of their subunits: p40 (common for IL-12 and IL-23), p35 (IL-12) and p19 (IL-23). Interestingly, while the expression of p35 and p19, similarly to other cytokines, was higher in DA animals, AO rats expressed more p40 mRNA. Nevertheless, DA rats made more p40 protein as well as IL-12. Taken together, our data suggest that the differential regulation of IL-12/IFN-γ and IL-23/IL-17 axes, and p40 in particular, in the inductive phase of EAE could be responsible for the discrepancy in susceptibility to EAE between these two strains. WS05-03 IL-18-independent IL-18Ra engagement is required for the development of autoimmunity I. Gutcher a , E. Urich a , M. Prinz b and B. Becher a a University Hospital Zurich, Zurich, Switzerland; b University of Göttingen, Göttingen, Germany IL-18 is considered to be a vital cofactor, together with IL-12, for the polarization of T H 1 cells. T H 1 cells have long been suspected to be the major pathogenic cell population in autoimmune diseases such as rheumatoid arthritis or multiple sclerosis. However, we and others could recently, firmly establish that in the animal models for RA and MS, IL-12 is not a disease promoting factor and that T H 1 polarized cells are not a prerequisite for disease development. We therefore wanted to determine the role of another T H 1-polarizing cytokine namely IL-18 and its receptor in the context of EAE. Surprisingly however, we discovered that mice deficient in IL-18Ra are EAE-resistant, suggesting the presence of an alternative ligand with encephalitogenic properties. We could establish that loss of IL-18Ra lesions an accessory leukocyte and not lymphocytes directly. We further demonstrate here that IL-18Ra signaling promotes the generation of IL-17producing CD4 + T cells, which are now considered to be the major pathogenic population in EAE as well as other autoimmune diseases. Our results establish that IL-18 and T H 1 development are dispensible for EAE pathogenesis, while IL-18Ra signaling by an as yet unidentified ligand (IL-18RL) is absolutely essential for the generation of encephalitogenic T H -17 cells. WS05-04 Antibodies to native myelin oligodendrocyte glycoprotein are critical for severe chronic experimental autoimmune encephalomyelitis and demyelination in mice and marmosets N. Heijmans a , A. Jagessar a , P. Smith a , T. Myelin oligodendrocyte glycoprotein (MOG) is a powerful encephalitogen for experimental autoimmune demyelination. However, the use of MOG peptides or recombinant proteins representing part of the protein, fail to address the possible pathogenic role of the full-length myelin-derived protein expressing post-translational modifications. Immunisation with central nervous system (CNS) tissues from wild-type (WT) and MOGdeficient (MOG −/− ) mice demonstrates that MOG in myelin is necessary for the development of chronic demyelinating experimental autoimmune encephalomyelitis (EAE) in mice and marmosets since myelin from WT mice induces chronic EAE while from MOG −/− myelin induced a mild selflimiting monophasic disease.Following immunisation all animals developed T-cell responses to recombinant mouse MOG (rmMOG) indicating that MOG released from myelin is antigenic. The lack of chronic disease following immunisation with MOG −/− myelin indicated that such responses were not pathogenic. Antibodies in WT but not MOG −/− myelin immunised animals recognised rmMOG and a MOG transfected fibroblast cell line but not MOG peptides. In all animals antibodies to myelin basic protein, oligodendrocyte specific protein, proteolipid protein and galactocerebroside were observed but did not correlate with disease severity. The data indicate that the conformational form of MOG is crucial for generation of pathogenic antibodies while glycosylation of MOG protein is not required for such antibody recognition.These data reveal that immunisation with the post-translational modified form of MOG in myelin promotes chronic autoimmune demyelinating neurological disease probably due to pathogenic antibodies to unmodified protein rather than linear epitopes or T cells. WS05-05 Real time imaging of autoreactive effector T cells in CNS lesions of Autoimmune Encephalomyelitis Experimental Autoimmune Encephalomyelitis (EAE) is induced by brain antigen-specific CD4 + T cells. Upon transfer to healthy recipients the cells induce massive CNS inflammation and severe paralytic disease. Using two-photon microscopy we visualize the behavior of retrovirally labeled MBP-specific T cells in the CNS of Lewis rats during different phases of clinical EAE. Imaging of intact animals and of acute spinal cord slices enabled us to track autoreactive effector T cells in meningeal/ perivasular areas as well as deep within the CNS parenchyma. We report here on two major findings: effector T cells redistribute in the course of EAE. In the primary clinical phase (days 2.5 after T cell transfer) the effector cells were restricted to perivascular locations. Beginning day 3 after transfer, however, they spread into the meninges and penetrated deeply into the CNS parenchyma. Their motility behavior profoundly changed over the course of EAE. T cell in the perivascular cuffs moved predominantly along the vessel walls. Cells within the CNS parenchyma moved seemingly non-directed. The cells displayed two distinct motility patterns: "motile" cells rapidly moved through the compact white matter, while "stationary" cells were attached and moved around a fixed anchor point. The ratio between these two fractions changed significantly over time. Whereas in early EAE the number of motile cells exceeded the one of the stationary cells this ratio became inverted during late EAE. During the last decade, we have witnessed unprecedented progress in the therapy of multiple sclerosis (MS). Despite the obvious benefit for clinical outcome, the new therapies have extensively tested the autoimmune concept of MS. Although evidence for autoimmune mechanisms in MS is largely indirect, proof can only be achieved by the demonstration that therapy based on the correction of immune deviation is able to restore neurological function. Interferon beta and Glatiramer, the two most widely used immunomodulating drugs in MS, are effective in reducing disease activity only by 30%. Does it mean that the autoimmune concept is challenged or do immune mechanisms in MS require additional points of interception? The clinical heterogeneity of MS may imply that several independent mechanisms exist and these might need to be separately targeted. In this regard, the use of nonantigen specific immune therapies aimed at a common pathway during the generation of the immune response might overcome some difficulties related to the claimed heterogeneity of MS. However, the recent history of side effects induced by Natalizumab have shown that antigen non-specific therapy can culminate in the severe loss of immune surveillance. Therefore, more selective immune therapies based upon the antigen reactivity of specifically-defined cell subpopulations or an effector immune mediator, may offer more promise. An additional point of complexity in MS therapy has been raised by the demonstration that neurodegeneration and brain atrophy represent important pathologic events of this disease. Currently, several immune-related molecules are being tested in clinical trials in MS and in the near future we should become more knowledgeable on how a correction of the immune response at different levels influences the course of the disease. WS06-01 Immune surveillance of the central nervous system in multiple sclerosis patients treated with natalizumab Objective: To evaluate whether treatment of multiple sclerosis (MS) patients with natalizumab, an antibody against VLA-4, interferes with immune surveillance of cerebrospinal fluid (CSF) and peripheral blood (PB). Background: Natalizumab therapy was recently associated with development of progressive multifocal leukoencephalopathy (PML), a demyelinating disorder of the CNS caused by JC virus (JCV) infection. Design/Methods: Cell numbers and cellular phenotypes in CSF and PB were analyzed by flow cytometry in MS patients treated with natalizumab, untreated MS patients, patients with other neurological disease (OND), and HIV-infected patients. Results: CSF total leukocyte counts, CD4 + and CD8 + T cells, CD19 + B cells and CD138 + plasma cells were significantly lower in natalizumab-treated MS patients compared with OND patients and untreated MS patients. Natalizumab therapy decreased the CD4:CD8 ratio in the CSF to levels similar to that of HIV-infected patients. Six months after cessation of therapy, low lymphocyte counts in the CSF persisted, whereas the CD4:CD8 ratio normalized. The patient with the highest total leukocyte, CD4 + and CD8 + T cell counts in the CSF experienced a clinical relapse. Interpretation: Our data suggest that a low CSF CD4:CD8 ratio in natalizumab-treated patients may confer an increased risk of developing PML. WS06-02 Successful therapy of multiple sclerosis (MS) targeting highaffinity IL-2 receptor reveals regulatory role of CD56 bright NK cells on T cell responses in humans Administration of Daclizumab, a humanized monoclonal antibody directed against the IL-2 receptor α-chain, strongly reduces brain inflammation in multiple sclerosis (MS) patients. The percentages and absolute numbers of immune cells were tracked by ex-vivo flow cytometry. Functional assays included flow-cytometry based proliferation and intracellular cytokine production; transwell T cell-survival assays and chromium-release cytotoxicity assays.Contrary to expectations, Daclizumab treatment lead to only a mild functional blockade of CD4 + T cells, the major candidate in MS pathogenesis. Instead, Daclizumab therapy was associated with a gradual decline in circulating CD4 + and CD8 + T cells and significant expansion of CD56 bright natural killer (NK) cells in vivo, and this effect correlated highly with the treatment response. In vitro studies showed that NK cells inhibited T cell survival in activated PBMC cultures by a contact-dependent mechanism. Moreover, CD56 bright NK cells isolated from Daclizumabtreated patients were directly cytotoxic to activated, but not resting autologous T cells in perforin-dependent manner. Positive correlations between expansion of CD56 bright NK cells and contractions of CD4 + and CD8 + T cell numbers in individual patients in vivo provides supporting evidence for NK-cell mediated negative immunoregulation of activated T cells during Daclizumab therapy. Our data support existence of an immunoregulatory pathway wherein activated CD56 bright NK cells inhibit T cell survival. This immunoregulation has potential importance for the treatment of autoimmune diseases, transplant rejection and toward modification of tumor immunity. WS06-03 Increased apoptotic elimination of activated T-cells following Campath-1H as a treatment of multiple sclerosis Multiple sclerosis (MS) is believed to be an autoimmune disease in which aberrant immune responses lead to demyelination, loss of oligodendrocytes and acute axonal transection. Suggested mechanisms for the breakdown in immune self-tolerance include impaired CD4 + CD25 hi function and the dysregulated apoptotic elimination of autoreactive Tcells.Campath-1H is a monoclonal antibody that induces T-cell lymphopaenia and reduces the MS relapse rate by 90%. By flow cytometry we investigated the effect of Campath-1H on the apoptotic death of T-cells emerging into the lymphopaenic environment. We observed a significant increase in spontaneous T-cell death and anti-Fas induced T-cell death (5% to 30%, and 10% to 50% respectively). Pancaspase inhibition largely reversed this, confirming cell death was predominantly apoptotic. Increased T-cell death was also observed following stimulation with myelin basic protein (MBP) and other specific antigens. Following treatment, however, stimulation with MBP and other recall antigens led to a reduction in cell death at day 3, when compared to unstimulated cells or to those exposed to neo-antigen (keyhole limpet haemocyanin). This implies that factors released early in the memory response of peripheral blood mononuclear cells extracted from a lymphopaenic environment protect against apoptosis. Mechanisms leading to the altered apoptotic state of T-cells and their altered response to MBP following Campath-1H are discussed.Elimination of activated T-cells is important in immune homeostasis and in the avoidance of immunopathology. Our findings suggest that Campath-1H's efficacy may, in part, be due to increased apoptotic elimination of activated T-cells. WS06-04 Frequency of CD4 + Foxp3 + T regulatory cells (Tregs) in peripheral blood of multiple sclerosis: Comparison to studies identifying Tregs as CD4 + CD25 high cells Multiple sclerosis (MS) is assumed to result from the breakdown of peripheral tolerance. Since CD4 + Foxp3 + T regulatory cells (Tregs) play roles in maintaining peripheral tolerance, it is important to determine if dysregulation of Tregs contributes to the onset and/or progression of MS. Although previous studies identified human Treg as CD4 + CD25 high cells, activated T cells also express CD25 molecule. To specifically assess the frequency of Tregs, we analyzed expression of Foxp3 by CD4 + CD25 + cells. Methods: PBMC were obtained from 19 consenting patients with MS and 13 healthy controls (HC). Among the patients enrolled, 11 patients were receiving IFN-beta therapy. Cells were stained with anti-human CD4 PE-Cy5 (RPA-T4) and anti-human CD25 PE (BC9), and then anti-Foxp3 FITC (PCH101) intracellularly. Expression of these molecules was determined by FACSCalibur. Results: CD4 + CD25 + cells were divided into CD25 low and CD25 high fractions. In HC samples, an average of 85% of CD4 + CD25 high expressed Foxp3, but a considerable number of Foxp3 + cells were also found among CD4 + CD25 low . In contrast, Foxp3-negative non-Tregs constituted more than 50% of CD4 + CD25 high in five patients on IFN-beta therapy and in one not receiving IFN-beta therapy. The proportion of Tregs in CD4 T cells in MS-PB was significantly decreased compared with that of HC-PB (2.83% vs. 5.51%, p < 0.05, t-test). Interestingly, IFN-beta therapy significantly increased the Treg frequency in MS-PB up to an average of 4.40% (p < 0.05, t-test). Discussion: We show a decrease in the frequency of circulating Tregs in MS. Of note, IFN-beta may partially reverse the deficit of Tregs in MS. WS06-05 Prognostic relevance of antimyelin antibodies for the progression to multiple sclerosis after a first demyelinating event: Results of the BENEFIT trial Patients with a clinically isolated syndrome (CIS) have a high risk of progression to clinically definite multiple sclerosis (CDMS). However, individual prognosis is unpredictable. Berger et al. reported a significantly increased risk of conversion to CDMS in patients with antibodies against myelin oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP) (NEJM 2003; 349:139-145) .In the BEtaferon (r) in Newly Emerging multiple sclerosis For Initial Treatment study (BENEFIT), a total of 468 patients with a CIS and at least two clinically silent brain MRI lesions were investigated. In this multicentre, double-blind trial, patients were randomised to IFNB-1b (n = 292) or placebo (n = 176) and treated for 24 months or until CDMS was diagnosed. Regular visits were scheduled for the collection of data on neurological disability and MRI before treatment and at study month 3, 6, 9, 12, 18 and 24. We measured serum anti-MOG and anti-MBP antibodies at baseline by Western blot.At month 24, 28% (IFNB-1b) and 45% (placebo) of the patients had reached CDMS. Data on the relation of antibodies to conversion to CDMS and "McDonald MS" will be presented.These analyses will allow an evaluation of the clinical relevance of anti-MOG/MBP antibody testing in a representative CIS population thoroughly characterized clinically and by MRI in the setting of a clinical trial. Autoimmunity to glycolipids is implicated in the pathogenesis of several neuropathic syndromes, in association with IgM monoclonal gammopathies, or following exposure to such bacteria as Campylobacter jejuni (Cj), that has cross-reactive lipopolysaccharides (LPS). A great deal has been learned about the anti-glycolipid antibodies, but relatively little is known about the role of T-cells in the associated neuropathies.Systemic transfer of IgG, but not of IgM antibodies, without T-cells, has been demonstrated to cause neurological dysfunction in experimental adult animals. IgMs might require the presence of T-cell reactivity, against the same or another nerve antigen, to exert their effects. Inflammatory T-cells could provide chemotactic factors or activate the monoclonal B-cells, facilitate IgM penetration through the blood nerve barrier, expose cryptic glycolipid antigens, or create a pro-inflammatory environment that amplifies the response.High titers of IgG anti-ganglioside antibodies are closely associated with variants of the Guillain-Barre syndrome. The antibodies are T-cell dependent as they are of the IgG1 and IgG3 isotypes. The T-cells might additionally damage the nerves via T-cell dependent effector mechanisms.LPS activates B-cells via T-cell independent mechanisms, so that the development of a T-cell response following Cj infection, is unexplained. However, the CD1 pathway of lipid antigen presentation has recently been linked to both GBS and CIDP, so that if Cj LPS is presented to T-cells via the same pathway, it might also induce a T-cell dependent anti-glycolipid response. The role of T-cells and of CD1 mediated antigen presentation warrant further investigation. Objective: Campylobacter jejuni (C. jejuni) is now the most recognized antecedent cause of Guillain-Barre syndrome (GBS). Recently, we identified and characterized the iron-binding protein Dps from C. jejuni (C-Dps). The aim of this study was to determine whether C-Dps contributes to the pathogenesis of GBS. Methods: We tried to evaluate the effect of exposure to C-Dps on nerve conduction after intraneural injection. Binding studies of C-Dps to rat neuronal tissues were performed. Furthermore, we evaluated the clustering of sodium channels at the node of Ranvier and the direct effect of C-Dps to sodium channels using the whole-cell patch-clamp method. Results: We found that (a) C-Dps, but neither PBS nor heat-denatured C-Dps, when injected into the rat sciatic nerve significantly decreased proximal-compound muscle action potential (CMAP), while motor conduction velocity did not change significantly, (b) C-Dps binds to the nodes of Ranvier and the outer surfaces of myelin sheath, (c) in the C-Dps injected sciatic nerve, immunostaining of the sodium channel was markedly decreased at the node of Ranvier, (d) C-Dps had no direct effect on sodium channels in the rat hippocampal CA1 neuron. Conclusions: C-Dps has direct neuro-toxicity mediated by the downregulation of sodium channels at node of Ranvier. WS07-02 Campylobacter jejuni cell body antigen induced experimental autoimmune neuritis C.-S. Koh a , S. Miyoshi a , K. Oana a , K. Nakayama a , T. Ehara a , M. Kyogashima b and T. Shin c a Shinshu University, Matsumoto, Japan; b Aichi Cancer Center Research Institute, Nagoya, Japan; c Cheju National University, Jeju, South KoreaThe etiology of the Guillain-Barré syndrome (GBS) still remains elusive. GBS is currently viewed as a group of syndromes with several distinctive subtypes. 15-40% of the patients with GBS develop the syndrome after being infected by the Gram-negative bacterium Campylobacter jejuni (C. jejuni), a leading cause of acute gastroenteritis in humans. Experimental autoimmune neuritis (EAN) is considered the in vivo model of GBS. There has been, however, no animal model of EAN which is induced by C. jejuni cell body antigen. We here report that a new form of EAN was successfully induced for the first time in animals by using C. jejuni cell body antigen. C. jejuni, Penner serotypes O:19, was isolated from the patient with GBS and cultured. EAN could be induced in Th1 bearing animals such as Lewis rats, SJL/J mice, and DBA/2 mice, but not be induced in Th2 bearing animals such as C57BL/6 mice by a single sensitization of C. jejuni cell body antigen in complete Freund's adjuvant. By histology inflammatory cell infiltration was observed in sciatic nerves, especially in perineurium, and cauda equine. Immunohistological study showed that infiltrated cells were ED1, a marker of macrophage, positive. Taken together, these findings suggest that Th1 immune responses may play an important role in the pathogenesis of the new form of EAN by using C. jejuni cell body antigen. WS07-03 The effect of rho-kinase inhibitor fasudil in experimental autoimmune neuritis Objective/methods: To study the potential role of rho-kinase inhibitor fasudil in the course of EAN (Experimental autoimmune neuritis), we induced EAN in Lewis rats using P0 peptide 180-199. In the preventive group, PBS (n = 14) and fasudil 100 mg/kg/day (n = 13), rats were immunized and PBS or fasudil were delivered via Alzet minipump starting day-2 until day 30 post-immunization. On Day 10, splenocytes were harvested, cultured and assessed for incorporation of [ 3 H] thymidine for proliferation assays. Cytokine assays (IFN-γ and IL-4) were also done. Weight and clinical scores were recorded daily. Fasudil was started at the onset of clinical signs (days 12-13) delivered continuously also via Alzet miniosmotic pump for 28 days. Results: In the preventive study, the incidence of EAN was 78% (11/14) in the PBS group while it was 38% (5/13) for fasudil and significantly different using Fisher's exact test (p = 0.054). The mean clinical scores were also significantly lower in the fasudil-preventive group than the PBS group using Mann-Whitney U-test (p < 0.05) on days 14-23 with days 21-22 (p value < 0.001) of EAN. Proliferation assays on Day 10 also showed significantly higher [ 3 H] thymidine incorporation in the PBS group than the fasudil-preventive group. While the IFN-γ of the culture supernatant also showed higher levels in the PBS group as well as higher IFN-γ/IL-4 ratio also for PBS. In the treatment study, mean clinical scores were also significantly lower in the fasudil-treatment group than the PBS group on days 21-25 post-immunization with a p value <0.05. Conclusion: Fasudil hydrochloride shows promise of effectiveness in promoting and probably hastening neurological recovery during the course of EAN. WS07-04 "Readthrough" acetylcholinesterase facilitates inflammation associated neuropathies in an antisense suppressible manner Inflammatory factors that penetrate through a disrupted blood-nerve barrier may play a common pathophysiological role in various neuropathies. Disruption of the blood-nerve barrier concomitant to induction of systemic inflammation by lipopolysaccharide (LPS) administration caused a transient conduction block in rats. LPS administration leads to proteolytic cleavage of the stress-induced "readthrough" acetylcholinesterase variant (AChE-R), and accumulation of its distinct cleavable C-terminal peptide (ARP). We therefore examined the involvement of AChE-R and ARP in inflammation-associated nerve conduction impairments. Dissociated rat splenocytes were reacted in vitro with Campylobacter jejuni (Cj) or E. coli LPS. Cell-free reactive splenocyte medium was injected intra-neuronally to rat sciatic nerves. Medium from untreated splenocytes served for control. Compound muscle action potentials recorded from the intrinsic foot muscles following stimulation proximal-and distal to the injection site were recorded, and the proximal-to-distal amplitude ratio (PDR) was calculated, representing conduction efficiency. Nerve segments that include the injection site were collected for analyses. AChE-R mRNA was identified by in situ hybridization within the cytoplasm of Schwann cell bodies, and myelin sheaths. Immunohistochemistry identified ARP within the cytoplasm of Schwann cells, and additionally within axons. Furthermore, systemic treatment with an antisense oligonucleotide, EN101, which suppresses AChE-R expression attenuated conduction block formation following intra-neural reactive splenocyte medium injection. Thus AChE-R and ARP may play a key role in inflammation-associated neuropathies, and EN101 might emerge as a novel treatment for such neuropathies. WS07-05 The role of membrane attack complex (MAC) in complement component C6 deficient Lewis rat The role of antibody deposition and complement activation, especially the role of membrane attack complex (MAC) in the mediation of injury in experimental allergic neuritis (EAN) is thought to be critical. In previous studies, we have examined the course of active experimental allergic encephalomyelitis (EAE) in a strain of PVG rats that are totally deficient in the C6 component of complement (PVG/C6 −/− ) thus unable to assemble MAC. These PVG/C6 −/− rats developed a less severe EAE compared with PVG/C rats following immunization with myelin basic protein (MBP). However, the role of complement in demyelination could not be examined because in the EAE model there is limited central nervous system demyelination. Furthermore, PVG rats are not susceptible to either active or passive EAN.We therefore backcrossed PVG/C6 −/− onto the Lewis rat, an EAN susceptible train. After 10 generations we have a congenic Lewis rat that is deficient in C6 (Lewis/C6 −/− ) that is unable to form MAC. Otherwise, these rats are healthy and immunologically identical to normal Lewis rats. This strain of congenic Lewis rat will allow us to directly examine the role of MAC in EAN.In this study we have shown that Lewis/C6 −/− can be susceptible to EAN, but with a lower disease severity as compare to the wild type Lewis rats. These results suggest that although MAC has a role in demyelination and the overall pathogenesis of the disease, but not essentially a sole factor to these processes. The roles of autoantibodies in central nervous system (CNS) diseases, such as multiple sclerosis, are not clear, but there is increasing evidence for specific autoantibodies to neuronal proteins as markers for other paraneoplastic and non-paraneoplastic neurological diseases. Onconeural antibodies are usually measured by indirect immunohistochemistry/ immunofluorescence but can also be measured by commercial immunoblotting tests. The antibodies (to Hu, Yo, Ri, Ma/Ta, CRMP5/CV2, Amphiphysin, Tr) are highly disease specific, are often found at very high titer, and usually indicate the presence of a particular tumour (e.g. small cell lung cancer, ovarian etc.). However, there are many unanswered questions concerning their relevance; for instance, most of the onconeural antibodies are not thought to be pathogenic and the associated diseases are likely caused by cytotoxic T cells. Whether these are controlled by specific regulatory cells is one question that needs to be answered. There are also several non-paraneoplastic conditions in which there are potentially causative antibodies. Voltage-gated potassium channel antibodies are associated with (usually non-paraneoplastic) peripheral and CNS disorders and are likely to be pathogenic, although this has not been proved for the CNS disorders. Glutamate receptor antibodies can activate the receptors and mimic excess glutamate, leading to neuronal loss. Enolase antibodies are being identified in several diseases (Hashimoto's, retinopathies) and may induce calcium release from internal stores. In addition, there are a number of other antibodies, and techniques employed to identify further specificities in serum and CSF, that should prove useful in the future. WS08-01 Autoantibodies against the amino-terminal of alpha-enolase in Hashimoto's encephalopathy M. Yoneda a , A. Fujii a , A. Ito a , H. Nakagawa a and M. Kuriyama a a University of Fukui, Fukui, Japan Background: Hashimoto's encephalopathy (HE) is an autoimmune encephalopathy distinct from myxoedema encephalopathy, associated with Hashimoto's thyroiditis (HT). The detection of anti-thyroid antibodies in patient sera is helpful but not sufficient for the diagnosis of HE because of the high prevalence in the normal population. Recently, we reported autoantibodies against the amino (NH 2 )-terminal region of alpha-enolase (NAE) as a useful diagnostic marker of HE. Methods: We performed immunoblotting analyses of patient's serum against recombinant NAE. We investigated the prevalence of anti-NAE autoantibodies in the sera from 25 patients with HE, and examined their clinical features, laboratory and MRI findings. Results: Patients with HT, who showed encephalopathy with the presence of anti-thyroid antibodies and steroid-responsiveness, demonstrated a high prevalence of anti-NAE autoantibodies in their sera from HE (68%, 17 of 25), compared to a low prevalence in HT without encephalopathy (10%, 2 of 20) (p < 0.001) and absence in other autoimmune conditions. Consciousness disturbance, seizures, cognitive impairment and EEG abnormalities were common. The patients with anti-NAE autoantibodies showed good steroid-responsiveness and a high prevalence of abnormality on EEG (100%).Conclusions: This study demonstrated a high prevalence of serum anti-NAE autoantibodies in patients with HE. Anti-NAE autoantibodies are emphasized, instead of anti-thyroid antibodies, as a useful serological diagnostic marker of HE, and should be included in the diagnostic criteria of HE. WS08-02 Examining pathogenicity of anti-enolase antibodies in paraneoplastic and autoimmune retinopathy Thimmappa Anekonda, Landon Karren, Richard G. Weleber, and Grazyna Adamus Neurological Sciences Institute, Casey Eye Institute, Oregon Health and Science University, Portland, Oregon, USA Anti-enolase autoantibodies have been associated with retinal degeneration in patients with autoimmune and paraneoplastic retinopathies. These patients usually suffer from a slow progression of central vision loss and central cone dysfunction, and show a significant reduction in their electroretinograms. There is considerable interest in examining the relationship between anti-enolase antibodies and retinal degeneration since their role in the pathogenicity of retinopathy is not fully understood. We characterized patients' sera for anti-enolase reactivities using Western blotting and ELISA. Investigations were performed according to the guidelines of the "Declaration of Helsinki" and informed consent was obtained at the time of blood collection.Five epitopes in the enolase protein were determined with the major epitope located within the amino acid sequence 36-41. Normal sera without anti-enolase reactivity did not react with enolase peptides. We propose that blocking enolase function by anti-enolase antibodies may lead to a depletion of cellular ATP, a subsequent increase of intracellular calcium, and finally to cell death. In this study, we found that anti-enolase antibodies significantly reduced the cytosolic ATP when retinal neurons were grown with anti-enolase antibody. In addition, by measuring the level of intracellular calcium in retinal cells treated with patients' anti-enolase IgGs, we found that IgGs were potent inducers of intracellular calcium increase. Normal IgG did not induce intracellular calcium or reduce cytosolic ATP. Using calcium blockers, glycolytic inhibition by anti-enolase antibodies led to a sudden release of endoplasmic-stored Ca 2+ . In conclusion, our results strongly suggest that anti-enolase antibodies have a potential role in the pathogenicity of retinal degeneration by deregulating glucose metabolism in retinal cells.This work was supported by grant from NIH EY13053, FFB, and RPB. WS08-03 Glutamate receptor antibodies induce brain damage and neurobehavioral dysfunction in 'autoimmune epilepsy' and 'neuropsychiatric SLE' Mia Levite, Yonatan Ganor The Weizmann Institute of Science, Rehovot, IsraelNow, after decades of documenting the massive brain pathology caused in numerous human and animal neuronal injuries/diseases, by excess glutamate, the time of glutamate-receptor autoantibodies has come.We can no longer ignore the growing incrementing evidences, showing that two types of glutamate-receptor autoantibodies: anti-AMPA subtype 3 (GluR3) and anti-NMDA R2A/B autoantibodies, are present in serum and/or CSF of patients with epilepsy, SLE and stroke, and are highly pathogenic to the CNS, by virtue of killing neurons and glia, inducing various neurobehavioral impairments, and evoking/ augmenting epilepsy. Importantly, in sharp contrast to the blocking autoantibodies playing a pathogenic role in most/all autoimmune diseases, glutamate-receptor antibodies are activating antibodies that can activate their antigen: a key neurotransmitter receptor. As such, glutamate-receptors antibodies can mimic glutamate or excess glutamate, activate ionotropic glutamatereceptors, trigger ion currents, kill neurons, and lead to CNS damage and dysfunction.Herein we wish to summarize the in vivo and in vitro evidences in humans, rabbits, rats and mice, revealed in our lab, as to the presence of glutamate-receptor autoantibodies in patients with epilepsy and SLE, their pathogenic activity, and their unique mechanism of action. Strikingly, the autoimmune-mediated brain damage caused by glutamate-receptor autoantibodies is different in every aspect from that occurring in MS, to date the most studied human autoimmune-mediated CNS pathology.We recommend that glutamate-receptor antibodies should be considered, from now onwards, as a genuine cause for autoimmune-mediated brain damage, unrecognized and underestimated thus far, and that we should develop means to arrest them. WS08-04 Monocloning oligoclonalityor unraveling the secret of oligoclonal bands: Resurrection of expanded CSF plasma clones as recombinant human monoclonal antibodies Neuroborreliosis is a chronic inflammatory disease of the central nervous system, caused by a tick-borne spirochete, Borrelia burgdorferii (Bb). While in neuroborreliosis and other chronic CNS infections oligoclonal CSF immunoglobulinswhich are detectable as so called oligoclonal Ig bands (OCB) after isoelectric focusingare directed against the causative infectious agent, the specificity and disease relevance of OCB in other chronic inflammatory diseases of the CNS such as multiple sclerosis are still unknown. We have established an experimental system to identify the antigen specificity of OCB, which are the product of oligoclonally expanded CSF plasma cells. Starting from single cell RT PCR of individual CSF plasma cells we identified expanded clones and expressed their heavy and light Ig chain genes as recombinant human monoclonal antibodies (mAb) in a eukaryotic expression system. Using immunoprecipitation of Bb lysate and subsequent MALDI analysis we could identify the antigen specificity of the derived mAb to be directed against the p41 antigen of Bb. We could show that the affinity of this patient derived human mAb is 1000 times higher to native Bb lysate than to denatured recombinant p41. Our results demonstrate on the one hand the validity of our experimental system in resurrecting the original antigen specificity of expanded CSF plasma cell clones, which can be instrumental in the elucidation of the antigen specificity of OCB in MS and related diseases. On the other hand our results imply, that OCB may be directed against conformation dependent epitopes, which might escape identification by currently popular approaches using recombinant antigen expression libraries. WS08-05 Cerebrospinal fluid proteomics profiling predicts cognitive impairment in HIV-1 infected Hispanic women HIV-1 associated cognitive impairment is a diagnosis of exclusion after searches for opportunistic infections and malignancies are eliminated. Thus, the search for disease biomarkers remains timely and relevant. In a step towards achieving this goal an integrated proteomics platform combining surface enhanced laser desorption ionization time of flight (SELDI-TOF), reverse-phase high performance liquid chromatography (RP-HPLC), one dimensional SDS-PAGE electrophoresis, and liquid chromatography tandem mass spectrometry (LC-MS/MS) were employed to determine links between cerebrospinal fluid (CSF) profiling and cognitive impairment. CSF was collected from 20 HIV-1-infected Puerto Rican Hispanic women and fractionated by RP-HPLC. Nine differentially expressed protein peaks were detected on CSF fractions by SELDI-TOF. Electrophoresis and LC MS/MS studies identified 14 common proteins. Among the proteins preferentially identified in cognitively impaired subjects were familial ALS mutant of Cu + 2 , Zn + 2 superoxide dismutase (mSOD, 15,839 mw), lymphocyte cytosolic protein 1 (L-plastin, 70,289 mw), migration inhibitory factor-related protein (MIF, 10,835 mw), Galectin-7 (GAL7, 14,944 mw), and VGF nerve growth factor (VGF, 67,287 mw). Western blot examination confirmed the preferential protein expression of SOD in cognitively impaired subjects in both Puerto Rican and North American cohorts. This study highlights the utility of proteomics platform profiling towards uncovering unique biomarkers for HIV-1-associated cognitive impairment.Supported by grants NIH-NINDS U54 NS4301, NIH-NCRR-RCMI-CRC-P20RR11126, and NIH-NCRR-RCMI G12 RR-03051 for the SELDI-TOF and Beckman 2000 Instruments. Experimental autoimmune encephalomyelitis (EAE) is an animal model of the human disease multiple sclerosis (MS). EAE is thought to be mediated by Th1 cells that secrete proinflammatory cytokines such as IFN-γ and lymphotoxin, although T cells that secrete IL-17 are also thought to play a role in disease pathogenesis. Given their central role in disease pathogenesis, T cells represent an excellent target for regulation and potential therapies. We have been interested in the pathways that regulate the development of inflammation in inflammatory central nervous system (CNS) diseases. In EAE, we have shown that silencing T-bet, a transcription factor important in Th1 cell differentiation, can suppress EAE development. We have also investigated members of the nuclear hormone receptor superfamily, such as peroxisome proliferator activated receptors (PPAR)-α, and their ability to regulate CNS inflammation. Using chromatin immunoprecipitation assays, we show that PPARα can bind the regulatory regions of the IL-4 and IL-5 genes and promote secretion of these cytokines. Using the PPARα agonist gemfibrozil, we can demonstrate receptor-dependent increases in GATA-3, IL-4 and IL-5 and inhibition of T-bet and IFN-γ. These data suggest that targeting the transcriptional regulation of T cell differentiation could be a successful therapeutic strategy in immune-mediated CNS inflammatory diseases. Objective: To investigate whether experimental autoimmune encephalomyelitis (EAE) protection by atorvastatin (AT) requires STAT6-mediated Th2 differentiation. Background: AT prevents and reverses EAE and is currently tested for multiple sclerosis treatment. Several EAE studies reported a Th2 differentiation of myelin reactive T cells under AT treatment with an upregulation of STAT6 signaling which controls Th2 differentiation. In order to elucidate the clinical significance of AT-mediated Th2 differentiation we evaluated AT-treatment in STAT6-knockout (−/−) mice. Methods: C57BL/6 STAT6 −/− mice were fed orally with 1 or 10 mg/kg/d AT starting either 2 days prior to immunization or after MOG p35-55 EAE was established. Mice treated for EAE prevention were evaluated for proliferation and cytokine secretion of MOG p35-55-specific T cells. Naïve T cells from B10.PL IL-4-GFP reporter mice were differentiated into Th2 or Th1 cells as distinguished by FACS (IL4-GFP → Th2). Resting Th1 and Th2 cells were stimulated with anti-CD3/anti-CD28 in the presence of AT. Results: AT treatment could effectively prevent and reverse EAE in STAT6 −/− mice comparable to its treatment effect in C57BL/6 wild-type mice. MOG p35-55-specific proliferation and secretion of IFN-g, TNF-a was reduced in T cells from AT-treated STAT6 −/− mice. In vitro, AT inhibited proliferation of both Th1 and Th2 cells in a dose-dependent manner. Conclusions: Our data indicate that AT can prevent and reverse EAE independent of STAT6-mediated Th2 differentiation. Other mechanisms besides Th2 differentiation, including the inhibitory effect on T cell proliferation and Th1 differentiation reported here significantly contribute to the therapeutic effect of AT in EAE. WS09-02 Oral administration of anti-CD3 antibody suppresses MOG induced autoimmune encephalomyelitis (EAE) in NOD mice, but not in NOD IFN-γ −/− or IL-10 −/− mice R. Maron, A. Tamvacakis and H.L. WeinerCenter for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115 USA One of the major goals for immunotherapy of autoimmune diseases such as multiple sclerosis (MS) is induction of regulatory T cells. We found that feeding anti-CD3 antibody suppresses PLP induced EAE in SJL mice via induction of regulatory T cells that function in a TGF-β dependent fashion (Nature Medicine, in press). IFN-γ −/− NOD mice developed severe EAE with an exacerbated disease pattern which was not ameliorated by oral anti CD-3. IL-10 −/− NOD mice developed a mild relapsing disease which was exacerbated by oral anti-CD3. IFN-γ −/− NOD mice secreted increased IL-2 and IL-6 and decreased TGF-β whereas IL-10 −/− NOD mice had decreased IL-6 and no increase in TGF-β. Oral anti-CD3 had no effect on MOG specific IFN-γ secretion in either NOD or IL-10 −/− NOD mice. These results demonstrate that EAE disease patterns in the NOD mice are affected by deficiencies in IFN and IL-10 and that the inability of oral anti-CD3 to suppress EAE appears linked to the inability to induce regulation by TGF-β in these mice. WS09-03 Therapeutic effect of mouse CD28 superagonist in murine EAE: Activation of pre-existing regulatory T cells F. Lühder a , F. Elias b , S. Schilling a , T. Hünig b and R. Gold a a Institute for Multiple Sclerosis Research, Göttingen, Germany; b Institute for Virology and Immunbiology, Würzburg, Germany CD4 + CD25 + regulatory T cells (Treg cells) are crucial for controlling autoimmunity and inflammation. Previously we could show that a rat CD28 specific superagonistic monoclonal antibody is able to activate and expand Treg in vivo. This was considered the major mechanism for its capability to ameliorate EAE in a Lewis rat model. In the present study we demonstrate that a new, analogous superagonistic antibody specific for mouse CD28 significantly delayed disease onset and reduced disease severity of MOGinduced EAE in C57Bl/6 mice. The administration of the mAb ameliorated EAE both in a preventive and therapeutic setting. When CD4 + CD25 + Treg cells were depleted in vivo before superagonist administration the protective effect was abolished, demonstrating the importance of these cells as mechanistic link for the protective effect of the CD28 superagonist. In cases where the depletion of Treg was incomplete, however, the CD28 superagonist restored the CD4 + CD25 + FoxP3 + Treg compartment within 3 days to more than normal levels indicating its potency in Treg activation and expansion in vivo. Adoptive transfer experiments using lymphocytes either from GFP transgenic mice or labeled with CFSE revealed that the expansion of the Treg compartment was not due to conversion of CD4 + CD25 − T cells into CD4 + CD25 + Treg cells but rather the result of rapid proliferation of pre-existing Tregs.These results show that even under conditions of a partially depleted Treg compartment, as is associated with human autoimmune diseases, CD28 superagonist therapy can prevent the disease by activating and expanding regulatory T cells. WS09-04 Modulation of Notch signaling affects encephalitogenic potential of autoreactive T cells Background: Notch regulates differentiation of many cell types, including CD4 T cells. Interaction between distinct Notch receptors and their ligands: Jagged and Delta-like effects peripheral T cells responses. Objective: In our study we examined the effect of Notch pathway manipulation on proliferation, cytokine profile and encephalitogenic potential of autoreactive T cells. Methods: Lymph node cells (LNCs) isolated from SJL/J mice preimmunized with PLP 139-151 were cultured with gamma-secretase inhibitor (GSI) or with antibodies neutralizing distinct Notch receptors. ELISA and CFSE proliferation assay were used in order to measure cytokine production and proliferation of lymphocyte populations, respectively. The level of Notch protein and expression of Notch genes were assessed by immunoblotting and real-time PCR, respectively. After 3-day culture, LNCs were transferred via tail vein to normal SJL/J mice in order to induce experimental autoimmune encephalomyelis (EAE). The clinical course of EAE in all groups of mice was observed and histopathology of brain and spinal cords was performed. Results: GSI treatment decreases expression of all four Notch genes and reduces production of IFN-γ in PLP-stimulated autoreactive T cells. GSItreated lymph node cells induce milder disease than control LNCs. Histopathology of brains and spinal cords from the 'GSI-treated' mice shows diminished inflammation. In the blocking experiments, IFN-γ production was markedly decreased by anti-Notch3 Ab treatment, while it was not affected by selective Notch1 or Notch2 inhibition.Conclusions: Modulation of Notch signaling in autoreactive T cells affects their encephalitogenic potential. Notch may be a promising therapeutical target in autoimmune diseases, including MS. WS09-05 The role of T-bet in the regulation of inflammation in the CNS Petra D. Cravens, Anne R. Gocke, Sara C. Northrop, Li-Hong Ben, Rehana Z. Hussain, Michael K. Racke and Amy Lovett-Racke University of Texas Southwestern Medical Center, Department of Neurology and Center for Immunology, USA Our group has recently shown that suppression of the transcription factor T-bet (T-box expressed in T cells), with intravenously administered siRNA directed against T-bet prevented the onset of experimental autoimmune encephalomyelitis (EAE). Since EAE is an immune-mediated, demyelinating disease of the central nervous system that serves as an animal model for multiple sclerosis, it would be appropriate to determine whether silencing of T-bet using siRNA in mice with ongoing EAE would be beneficial. SiRNA-T-bet or siRNA-Nonsense (siRNA-NS) were administered intravenously at 2 timepoints to B10.PL mice that had developed EAE after adoptive transfer of encephalitogenic T cells. Splenocytes isolated from mice treated with siRNA T-bet produced less IFN-γ, had reduced IL-23R expression than mice treated with siRNA-NS. To examine genes that may be trans-activated by T-bet in vivo, chromatin immunoprecipitation assays were performed. T-bet bound to the IL-23R promoter but not the CXCR3 promoter. Engagement of the IL-23R on IL-23R + T cells results in IL-17 secretion and IL-17 expression in the CNS of siRNA-T-bet mice was decreased. Examination of immune cells present in the CNS of these mice by multi-parameter flow cytometry indicate that both IL-17 and IFN-γ may be produced by CD45 + CD4 + CNS cells. The percentage of T cells able to release IFN-γ and IL-17 are reduced in mice treated with si-RNA T-bet. There has been longstanding interest in how B cell responses may contribute to the pathology of neurological diseases. Traditionally, the premise has been that any such contribution relates to the potential of B cells to produce auto-pathogenic antibodies. Targeting autoantibodies continues to be an important therapeutic approach, particularly in disorders where the role of antibodies is well established, such as in myasthenia gravis. In other conditions such as multiple sclerosis, the role of circulating antibodies has been less clear, though pathologic studies continue to implicate CNS-reactive antibodies, as well as B cells within the CNS compartment. Recently, new insights into fundamental properties of B cells have suggested that these cells may contribute in an antibody-independent fashion, both to normal immune responses, as well as in the context of immune mediated diseases. This session and the associated posters will consider the roles of humoral immune responses, as well as antibody-independent B cell involvement in several neurological disorders. Also covered will be consideration of the roles and therapeutic mechanisms of action of existing therapies such as IVIG, as well as emerging approaches in which B cells are targeted more selectively. WS10-01 A disease-specific anti-IgG fraction isolated from pooled human IgG (IVIG) suppresses experimental myastehenia gravis (EAMG) Intravenous immunoglobulin (IVIG) administration has been beneficially used in recent years for the treatment of a variety of autoimmune diseases including myasthenia gravis. However, the mechanism of action of IVIG treatment and the fraction responsible for its therapeutic effect in autoimmune diseases are still not established. We have been studying the therapeutic effects of IVIG administration in experimental autoimmune myasthenia gravis (EAMG), a rat model for myasthenia gravis. The mechanism by which IVIG modulates EAMG involves suppression of Th1 cells and B cell proliferation but probably does not act via regulatory T cells.After establishing the optimal conditions for EAMG suppression by IVIG administrations we have employed this model in an attempt to isolate a specific suppressive fraction from IVIG preparation and to identify its therapeutic activity. We have demonstrated that chromatography of human IVIG on immobilized rat anti-AChR IgG, isolated from rats with EAMG, results in a complete depletion of the suppressive activity of the IVIG. Moreover, the eluted immunoglobulin fraction that had been adsorbed onto the anti-AChR antibodies and comprises less than 1/100,000 of the IVIG preparation retains the immunosuppressive activity of IVIG. This study supports the notion that the therapeutic effect of IVIG is mediated by an antigen-specific anti-immunoglobulin activity (anti-idiotypic activity) present in pooled human immunoglobulin and raises the possibility that diseasespecific anti-idiotypic activity can be fractionated from IVIG and become a preferential reagent for therapeutic purposes. Objective: CIDP can improve after intravenous immunoglobulin (IVIg) treatment. Most patients need intermittent IVIg to maintain improvement. This study aims to identify factors especially in relation to long-term treatment and prognosis. Methods: Data were collected from all CIDP patients known at the Erasmus Medical Center, treated with IVIg and followed for at least 2 months. 50 clinical and laboratory parameters were analyzed for a possible relation with improvement (Rankin scale). Results: 64 males and 31 females were followed for a period ranging from 2.5 months to 20 years (median 4.0 years). 77/95 (81%) patients improved after IVIg. Patients with sensory-motor disturbances (p = 0.002 HR 3.2) and a relative short duration of weakness (p = 0.008; HR 2.6) had a higher chance to reach remission after discontinuation of IVIg. 10% needed IVIg for a period over 8.7 years (maximum over 20 years). Severe side-effects were not seen. Conclusion: Most patients need IVIg for a long period of time. Patients with prognostic factors indicating the necessity for long-term treatment may be consideredespecially due to the high costs of IVIgto switch treatment in an early stage of disease. WS10-03 Changes in axonal excitability following intravenous immunoglobulin infusions in patients with dysimmune demyelinating neuropathy Boerio Delphine 1,3 , Creange Alain 2 , Hogrel Jean-Yves 3 , Lefaucheur Jean-Pascal 1 1 Service de Physiologie -Explorations Fonctionnelles, CHU Henri Mondor, Créteil, France; 2 Service de Neurologie, CHU Henri Mondor, Créteil, France; 3 Institut de Myologie, GH Pitié-Salpêtrière, Paris, France Background: Dysimmune demyelinating neuropathies are associated with changes in nerve excitability resulting from alterations of axon membrane properties. Objectives: To investigate the effects of intravenous immunoglobulin (IVIgs) on nerve excitability in patients with multifocal motor neuropathy with conduction blocks (MMN) or chronic inflammatory demyelinating polyneuropathies (CIDP). Methods: 16 patients (9 MMN, 7 CIDP) were evaluated before and after IVIgs (0.4 g/kg/day for 5 consecutive days). Absolute (ARP), relative refractory period (RRP) durations and percentages of refractoriness at 2 ms and supernormality at 7 ms interstimuli intervals were determined. Stimulus/response (S/R) and strength/duration (S/D) curves were established. Activity-dependent hyperpolarisation induced by voluntary contraction was appraised on the percentage of conduction block in affected territories. Results: Before IVIgs, patients had longer refractoriness and smaller supernormality than healthy subjects. IVIgs increased refractoriness (78.1 ± 5.8 vs. 69.0 ± 6.7%, p = 0.04) and prolonged RRP (3.1 ± 0.2 vs. 3.8 ± 0.3 ms, p = 0.05) but did not modify ARP duration and supernormality. The SD time constant decreased (242.7 ± 35.6 vs. 352.7 ± 45.2 μs, p = 0.04), while rheobase and S/R curve slope remained unchanged. IVIgs reduced the percentage of activity-dependent conduction block (11.5 ± 0.7 vs.− 5.6 ± 0.6%, p = 0.002). Conclusion: This electrophysiological study provides additional information to usual conduction studies. The refractoriness prolongation is consistent with changes in Na + conductance or nodal membrane properties, suggesting a reduction in intraaxonal Na + accumulation, decreasing the occurrence of hyperpolarisation. While the reduction of percentage of conduction block traduced an improvement in Na + /K + ATPase pump function and a greater conduction. Objective: To examine whether (1) a novel B cell regulatory cytokine network is influenced by the local Th1 or Th2 environment in patients with MS and controls (2) such modulation can be targeted therapeutically. Methods: Circulating CD19 + B cells were purified from untreated MS patients (Poser criteria) and matched healthy controls, then stimulated with B cell receptor engagement followed by CD40 stimulation. Modulation of B cell responses by the local T cell environment was assessed by addition of IFNγ (Th1) or IL-4 (Th2). B cell proliferation (thymidine incorporation) and TNFα, LT and IL-10 (ELISA) were subsequently measured. Results: B cell proliferation was no different between MS and controls, under all stimulation conditions. IL-4 addition generally enhanced B cell cytokine production, as expected, with no differences in TNFα production between MS and controls. Upon addition of IFNγ, MS patient B cells produced significantly more TNFα and LT compared to control B cells; IL-10 production was not different. Interestingly, following mitoxantrone therapy in MS patients, the addition of IFNγ during MS B cell stimulation was now associated with significantly less TNFα and LT induction compared to baseline data.Conclusion: When activated, MS patient B cells may abnormally enhance a pro-inflammatory environment by increased release of TNFα and LT. Therapy with mitoxantrone appears to reverse this abnormality, with a pattern suggesting that different B cell populations may contribute distinct cytokines to a local inflammatory environment. These insights are pertinent given the growing interest in B cell roles in MS. WS10-05 Lipid microdomain-mediated signaling: Implications for anti-MOG mediated demyelinating disease Antibodies to myelin components are routinely detected in multiple sclerosis (MS) patients. We find that antibody cross-linking in oligodendrocytes (OLs) of myelin proteins MOG or MAG results in (a) their rapid repartitioning into glycosphingolipid-cholesterol microdomains ('lipid rafts' 1,2 , small (nm) membrane entities with few proteins, which upon ligand-or antibody-mediated cross-linking can coalesce into functional signal transduction "activation centers"; high cholesterol/glycosphingolipid content in OLs/myelin suggested that rafts contribute functionally to their physiology. ), followed by (b) raft-dependent phosphorylation-dephosphorylation of specific proteins and downstream events, and in the case of MOG, (c) dramatic, rapid loss of myelin-like membrane. 3 Immunization of C57BL/ 6 mice with either rat or human MOG produces comparable anti-MOG ELISA titers, however, only human MOG yields B cell dependent EAE and is encephalitogenic in primed B cell deficient mice. Substituting Pro42 with Ser in human MOG (as in rat MOG) eliminates this B cell requirement; nonpathogenic IgGs bind recombinant mouse MOG and deglycosylated MOG in myelin, but only pathogenic IgGs bind glycosylated MOG; only purified IgG to human MOG binds to live rodent OLs in culture and, after crosslinking, induce repartitioning of MOG into lipid rafts and dramatic changes in cell morphology. 3 These data provide a strong link between in vivo and in vitro observations regarding demyelinating disease, further suggesting a biochemical mechanism for anti-MOG-induced demyelination, and suggest in vitro tools for determining autoimmune Ab pathogenicity in MS. 1 Kim and Pfeiffer (1999); Taylor et al. (2004 ), Schafer et al. NMSS-FG1423A(CM)/RG2394(NHR); NIH-NS10861/NS41078(SP).Workshop 11: Studies in neuroimmune pharmacology Studies in the growing inter-discipline of neuroimmune pharmacology R. M. Donahoe a and T. J. Rogers b a University of Utah, Salt Lake City, UT, USA; b Temple University, Philadelphia, PA, USA NeuroImmune Pharmacology is a 'new" interdisciplinary area of research now represented by the Society of NeuroImmune Pharmacology. This symposium, "Studies in Neuroimmune Pharmacology", represents some of the broad interests of this 'new' interdiscipline. These data have considerable implication for opiate addicts infected with HIV1. Data are also presented that show that mu opiate-receptor ligands induce desensitization of the chemokine receptor, CCR5, on macrophages via phoshorylation of protein kinase-C. These fundamental data are important to the role of opiates in neuroimmune function and HIV/AIDS. Similarly, the signal transduction mechanisms involved in stimulation of lymphocytes through delta-opioid T-cell surface receptors are presented, which further elaborates how opioids interact directly with cells of the immune system at the basic molecular level. A human immune-reconstituted mouse model is also used to show that the anti-HIV drug, indinavir, can be delivered effectively via a nanoparticle packaged into bone-marrow derived macrophages as drug carriers. This novel model offers new approaches and new hope for treatment of HIV/AIDS. Finally, structural MRI, magnetic resonance spectroscopy, and positron emission tomography studies were used in humans to show neurological changes in HIV1-infected subjects who also use cocaine and methamphetamines. These combined data show the promise of neuroimmune pharmacology to advance understanding of major medical problems and the means to their resolution. WS11-01 Cross-talk between the mu opioid receptor and CCR5 is mediated by protein kinase c zeta (PKCζ) R.T. Rahim and T. J. Rogers Center for Substance Abuse Research, and Fels Institute of Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA Previously, our laboratory has shown that heterologous desensitization of CCR5 is induced upon activation of the mu opioid receptor (MOR). The present study examined the signaling events mediating the inactivation of CCR5 by pre-treatment with [D-Ala-N-Me-Phe4-Gly-ol] enkephalin (DAMGO), a MOR-selective agonist. Cell lines, stably-transfected with MOR and CCR5, and human monocyte-derived macrophages were treated with DAMGO (10 − 6 M) and used in various assays. Co-immunoprecipitation and Western blot analyses showed that PKCζ associates with CCR5 and that DAMGO induced phosphorylation of PDK-1 (phosphoinositide-dependent kinase-1), a kinase that is known to activate PKCζ. In addition, DAMGO augmented the kinase activity of PKCζ observed upon immuno-precipitation of CCR5 and induced phosphorylation of CCR5. Cells transfected with PKCζ siRNA showed a significant inhibition of PKCζ kinase activity associated with CCR5. In further studies, desensitization of CCR5 led to inhibition of calcium mobilization and decreased chemotactic response to Mip1-beta. Use of a PKCζ pseudosubstrate inhibitor reversed these results. Overall, the data suggest that DAMGO-induced desensitization of CCR5 leads to phosphorylation of CCR5 and that PKCζ mediates this effect. Results of a nearly 5-yr follow-up study confirm this original observation. Male rhesus macaques at around 4 yr of age were adapted over a 1-1/2-yr period to a single-cage holding environment with psychological enrichments. Baseline data were collected, followed by introduction of an opiate-dependency paradigm (4 daily, 6-h apart, sc/im injections of morphine sulphate, 3-2 mg/kg/monkey). Two weeks after initiation of these injections, all monkeys were injected iv with 10,000 TCID 50 of SIVsmm9. Progression of infection and disease was followed for 4 yr thereafter. Morphine retarded progression of AIDS overall (P V 0.05), being largely selective for monkeys that progressed relatively rapidly to AIDS. Opiatedependency also altered cerebral spinal inflammation. Retardation of disease progression corresponded with an ability of opiates to reverse the negative correlation typically seen as viral load increases over time in conjunction with CD4 + T-cell loss. These data represent the most solid evidence to date that opiates alter AIDS progression. In view of conflicting data that opiates exacerbate AIDS progression, current understanding of this issue supports our prior conjecture that the influence of opiates on AIDS is conditionalbeing dependent on variable drugdependency, viral and immune states, and possibly relating to the influence of these factors over stress. Such findings and conclusions support a need for more thorough understanding of how opiates affect AIDS progression in drug-abusing populations. Opioids modulate an array of functional responses by T-cells, including proliferation, cytokine production, chemotaxis and expression of human immunodeficiency virus-1 (HIV). However, the signaling pathways mediating these effects of opioids have been largely unknown. Recently, we observed that delta opioid ligands exert dual effects on specific T-cell signaling cascades; as such, the opioid itself stimulates, whereas opioid pretreatment attenuates the signaling response to another agent. For example, brief exposure (15 min) to a specific delta opioid agonist (i.e., DADLE) dose-dependently induced phosphorylation of c-jun by activating phosphoinositide 3-kinase (PI3K), protein kinase B (Akt) and the mitogen-activated protein kinase (MAPK) c-jun NH2-terminal kinase (JNK). Moreover, DADLE stimulated the phosphorylation of activating transcription factor-2 (ATF-2; implicated in cytokine gene transcription) and its association with JNK. In contrast to such direct activation of PI3K/Akt-dependent signaling, pretreatment with DADLE inhibited Akt phosphorylation induced by stromal cell-derived factor-1 (SDF-1). Pretreatment of highly purified human peripheral blood T-cells with DADLE for 1 or 3 h, significantly reduced SDF-1-induced Akt phosphorylation by approximately 60% (n = 7). In contrast, DADLE failed to stimulate phosphorylation of the MAPKs, ERK1 and 2, and did not affect SDF-1-induced ERK phosphorylation. Recent studies also demonstrate that morphine inhibited Akt phosphorylation stimulated by SDF-1. Together, these studies demonstrate that endogenous and exogenous opioids can exert both direct and indirect modulatory effects on signaling cascades that depend on PI3K/Akt. WS11-04 Pharmacokinetic, immune, and anti-viral responses of nanoparticle indinavir in a murine model of HIV-1 encephalitis Howard E. Gendelman, MD; Center for neurovirology and Neurodegenerative Disorders 5880 Nebraska Medical Center; Omaha, Ne 68198-5880; Phone 402-559-8920; FAX 402-559-89922; email: hegendel@unmc.edu. ABSTRACT The introduction of highly active antiretroviral therapy for human immunodeficiency virus infection has all but revolutionized pharmacologic management of human disease. Nonetheless, complex dosing regimens, costs, untoward side effects, limited bio distribution and variable drug pharmacokinetic patterns have affected long-term use and notably, clinical efficacy, within the nervous system. We posit that nanotechnology-derived cell based systems may overcome such limitations. In a first step towards testing this idea, we developed a nanoparticle indinavir (NP-IDV) formulation packaged into bone-marrow-derived-macrophages (BMM) as drug carriers. Drug distribution and disease outcomes were assessed in immune competent and in non-obese diabetic severe combined immunodeficient mice (NOD/SCID) reconstituted with human peripheral blood lymphocytes. In the model, NP-IDV contained within BMM was adoptively transferred into mice. After a single administration, single photon emission computed tomography and histology demonstrated robust lung, liver, and spleen BMM distributions. Tissue and sera IDV levels were z50 nM/ml up to 2 weeks of observation. NP-IDV-BMM administered to virus-challenged immunodeficient humanized mice showed reduced numbers of virusinfected cells in plasma, lymph nodes, spleen, liver, and lung. Numbers of CD4 + T cells were restored after NP-IDV-BMM administration. NOD/SCID mice with active HIV-1 encephalitis showed robust delivery of NP-IDV to affect brain subregions. We conclude that a single dose of NP-IDV using BMM as a carrier is bio-available and effective and warrants consideration for human testing. WS11-05 Basal ganglia abnormalities in HIV patients and chronic psychostimulant drug users L. Chang, T. Ernst University of Hawaii, Honolulu, USA A variety of in vivo physiological neuroimaging techniques can quantitatively assess brain injury associated with HIV and/or substance abuse. Structural MRI demonstrates atrophy of caudate and putamen in subjects infected with HIV, but enlarged striatal volumes in those with chronic psychostimulant abuse (methamphetamine, cocaine, etc.). Hence HIV and psychostimulant use have opposite effects on brain volumes. Conversely, magnetic resonance spectroscopy (MRS) shows metabolite abnormalities that are additive in HIV patients and methamphetamine users. These spectroscopic abnormalities include decreased neuronal marker N-acetylasparate and elevated glial marker myoinositol, most prominently in the basal ganglia and frontal cortex. Since psychomotor slowing and attention deficits are major features of HIV dementia and psychostimulant abuse, dopamine dysfunction has been postulated. Dopaminergic terminals and synapses have the highest density in the basal ganglia regions. Positron emission tomography studies (with C-11 cocaine) indeed illustrate lower dopamine transporters in HIV patients with dementia, as well as individuals with chronic methamphetamine dependence, compared to seronegative non-drug users. Correlations between cognitive performance and dopamine transporter levels document the functional consequences of dopaminergic deficits. These findings suggest that methamphetamine and cocaine abuse further exacerbate brain injury in HIV patients. Therefore, dopamine augmentation might be beneficial in the treatment of HIV dementia. Collectively, findings from these neuroimaging studies provide insights into the pathophysiological mechanisms of HIV-associated brain injury and drug abuse, and might lead to new therapeutic approaches.Acknowledgments: Studies and resources supported by the NIH (NINDS, NIMH, NIDA, NCRR), the Department of Energy, and the ONDCP. WS11-06 The cholinergic anti-inflammatory pathway Lulloa@nshs.edu We recently discovered that the vagus nerve can control peripheral innate immune responses through a mechanism that can be employed for the treatment of inflammatory disorders [Nat Rev DD 4:673, 2005] . Electrical stimulation of the vagus nerve attenuates systemic inflammation in experimental sepsis by inhibiting the production of pro-inflammatory cytokines in the spleen through a mechanism dependent on the common celiac branch. Vagus nerve stimulation fails to attenuate endotoxininduced serum TNF levels in splenectomized animals. Acetylcholine, the principal neurotransmitter of the vagus nerve, inhibits endotoxin-induced TNF and HMGB1 release from human macrophages through a mechanism dependent on the α7-nicotinic acetylcholine receptor (α7nAChr). In vivo, vagus nerve stimulation reduces splenic TNF protein and mRNA levels in wild-type mice, but not in α7nAChr-knockout mice. Nicotine, a more selective cholinergic agonist, abolished HMGB1 release in a dose-dependent manner, and it was a more efficient inhibitor of macrophages. In vivo, treatment with nicotine attenuated serum HMGB1 levels, prevented lethal endotoxemia, and improved survival in established polymicrobial peritonitis, even when the treatment was started after the appearance of the clinical signs of sepsis. Nicotine has already been used in clinical trials for inflammatory disorders such as ulcerative colitis, but its clinical potential is limited by its collateral toxicity. Similar to the development of selective agonists for adrenergic receptors, selective nicotinic agonists for the α7nAChR may represent a promising pharmacological strategy for infectious and inflammatory diseases. Three broad questions have driven research into the immune response to HTLV-1. First, how does HTLV-1 persist in the individual host? In particular, what is the role of the immune response in controlling or limiting viral persistence? Second, why do some HTLV-1-infected people develop a consequent disease such as HAM/TSP or leukaemia, whereas the majority remain asymptomatic carriers of the virus? Is this difference in the outcome of infection due primarily to variation in the host or variation in the virus? Third, how is the inflammatory lesion in HAM/TSP initiated and maintained, and how can the inflammation be halted? Evidence on the immune response to HTLV-1 will be summarized from recent work in host and viral genetics, DNA expression microarrays, T-cell function and cell biology. This evidence favours two main conclusions: first, that the efficiency of a person's cytotoxic T-lymphocyte (CTL) response to HTLV-1 plays a dominant role in determining that person's proviral load of HTLV-1 and the risk of the associated inflammatory diseases; second, that HTLV-1 is persistently transcriptionally active and spreads directly between lymphocytes by a specialized cell-cell contact known as the virological synapse. Until recently, the inference that HTLV-1 is persistently transcribed rested entirely on evidence from in vitro experiments. However, new evidence from quantification of lymphocyte turnover in vivo strongly supports the view that there is persistent transcription of HTLV-1. These conclusions have implications for therapeutic approaches both in the inflammatory diseases and in the HTLV-1-associated syndrome of adult T-cell leukaemia, and in the immune response to persistent viral infections. WS12-01 Central nervous system (CNS) involvement by herpes simplex virus 1 (HSV 1) is restricted in C57BL/6 (BL/6) mice Following mucosal inoculation with HSV 1, susceptible strains of mice develop recurrent multifocal CNS demyelination preceded by the spread of virus from the lip, through the peripheral nervous system and to the CNS. In contrast, BL/6 mice do not develop CNS demyelination and viral access to the CNS is restricted.The objective of this study is to identify the immune mechanism(s) that restrict HSV 1 entry to the CNS of BL/6 mice. Controls along with BL/6 mice depleted of immune cell subsets and knock out (KO) mice were lip inoculated with HSV 1 strain 2. Viral titers in the lip, trigeminal ganglia (Tg), and brain were determined in serial studies.HSV 1 is restricted to the lip and Tg of controls and BL/6 mice treated with either α-NK1.1 monoclonal antibody (mAb), α-CD4 mAb, α-CD8 mAb, or a combination of α-CD4 and α-CD8 mAbs. In contrast, virus can spread throughout the CNS of B6.129P2B2m tm1unc /J mice with infection limited to the first 12 days post infection (PI).Thus γδ T-cells, NK/NKT cells, CD4 T-cells, and CD8 T-cells limit HSV 1 infection in the lip and TG but do not restrict the spread of virus in the CNS. In contrast, virus spreads throughout the brain in B6.129P2-B2m tm1unc /J mice (CD8 T-cells and NK/NKT cells affected) without significant mortality. This suggests that an interaction between innate and adaptive immunity is required to restrict the spread of HSV 1 to the CNS. WS12-02 In vivo selection of T-cell receptor junctional region sequences by HLA-DRB1 ⁎ 0101 human T-cell lymphotropic virus type 1 (HTLV-1) envelope gp21 peptide complexes in HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) Human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurological disease observed only in 1-2% of HTLV-1 infected individuals. We have previously reported that HLA-DRB1 ⁎ 0101 was associated with susceptibility to HAM/TSP in two independent HTLV-I infected cohort in Southern Japan (Kagoshima) and Northeastern Iran (Mashhad), whereas HTLV-1 envelope gp21-specific T cells were restricted by DRB1 ⁎ 0101. To further characterize the role of DRB1 ⁎ 0101 restricted Env specific CD4 + T cells in chronic HTLV-1 infection, we have exploited tetrameric MHC-peptide complexes along with magnetic cell sorting to purify HTLV-1 envelope gp21-specific CD4 + T cells directly from peripheral blood lymphocytes from two HLA-DRB1 ⁎ 0101 positive HAM/TSP patients. RT-PCR, spectratyping and sequencing analysis of TCR Vβs revealed that the Vβ-Dβ-Jβ junctional sequences derived from two unrelated HAM/TSP patients shared identical amino acid motifs in CDR3 region. Our data provide strong evidence that several different T cells bearing distinct TCRs are stimulated with HLA-DRB1 ⁎ 0101/envelope gp21 complexes and then proliferate in vivo, i.e. there is selection for certain TCR gene sequences exerted in vivo by the MHC-peptide complex. WS12-03 Safety and efficacy of interferon-α in 167 patients with HTLV-1-associated myelopathy (HAM)Objective: A post-marketing surveillance study was undertaken to investigate the safety and efficacy of interferon-α for HAM under routine treatment conditions. Methods: A total of 273 cases from 91 medical institutions were registered into the survey. Results: The rate of response to the treatment (rated as improved and fair) at 4 weeks was 67.1%. Factors which significantly affected efficacy at 4 weeks was initial Osame's motor disability score (OMDS) before interferon-α therapy, duration and stage of illness. Sustained improvement of OMDS for at least 5 months after stopping interferon-α was observed in 11 of 30 patients (36.7%). A total of 536 adverse drug reactions (ADRs) occurred in 146 patients, 46 of which were serious. The incidence of ADRs was higher than that of a previously reported controlled trial, although no specific ADRs occurred in patients with HAM. Conclusions: These results indicate that interferon-α treatment is effective for at least patients in the incipient stage of HAM, though the incidence of ADRs is rather high. Long-term administration of interferon-α may have some advantage in maintaining the improved condition during treatment. Thus, the most suitable protocol for interferon-α administration in HAM needs to be defined in the future. WS12-04 What enters the brain stays in the brain Neuro-AIDS is a complication of HIV infection affecting 1/3 of HIV+ individuals, triggered by the early presence of virus in the brain. Neurophysiological alterations allow recognition of early signs of CNS dysfunction. The analysis of alterations on immune-cells in the brain in correlation with CNS dysfunction, show a remarkable 5-15-fold increase of highly activated memory CD3 + CD8 + CTLs in infected brains. The specificity of these cells was investigated. In the blood and CSF, Tat-specific cells peak during the acute phase, and become undetectable thereafter. However, in the brain, these cells remain as a large proportion of accumulating CTLs. We hypothesize that virusspecific cells enter the brain during acute phase, and are maintained to chronic stages. At 28 dpi, the enrichment of Tat and Gag-specific cells in the brain was already observed, suggesting an early entry. Factors favoring CD8 survival, such as IL2, IL7 and IL15, were examined. IL2 and IL7 were undetectable, but IL15 increased by 3-5 fold in SIVinfected brains. In vitro, IL15 efficiently supported CD8 survival, increased Ki-67+ cycling cells, and those expressing memory markers. Our results suggest that IL15-rich brain environment supports survival of SIV-specific and bystander memory CTLs. Taken together, increased IL15 levels in infected brains favor activated pro-inflammatory virus-specific CTLs, which enter the brain during the acute phase, and persist, potentially participating on development of inflammatory pathology. WS12-05 Axonal transport of toxic lectin from the PNS targets inflammatory demyelinating lesions to sites of Wallerian degeneration in the CNS in Theiler's virus infection: Lesion development from the axon (inside) to the myelin (outside) I. Tsunoda a , T. Tanaka a , Y. Saijoh b , S. E. Doyle a , E. J. Terry a , and R. S. Fujinami a Departments of Neurology a , and Neurobiology and Anatomy b , University of Utah School of Medicine, Salt Lake City, Utah, USA In Theiler's murine encephalomyelitis virus (TMEV) infection, an animal model for multiple sclerosis (MS), axonal injury precedes inflammatory demyelination. The distribution of axonal injury during the early phase corresponds to regions where subsequent demyelination occurs during the chronic phase. We hypothesized that axonal injury recruits inflammatory cells into sites of Wallerian degeneration, leading to demyelination. We have established a method for induction of Wallerian degeneration in the central nervous system (CNS). This approach uses toxic lectin, Ricinus communis agglutinin (RCA) I, and does not lead to a blood-brain barrier breakdown. The lectin is injected into the sciatic nerve and is transported axonally, leading to the death of dorsal root ganglion cells and Wallerian degeneration of the posterior funiculus in the spinal cord. Using a microinjection system, we injected lectin into the sciatic nerve of SJL/J mice, 3 weeks after TMEV infection. Neuropathology was examined, 1 week after lectin injection. Control mice, which received TMEV but no lectin, had inflammatory demyelinating lesions only in the anterior and lateral funiculi. In contrast, lectin-injected infected mice had lesions in the anterior and lateral funiculi as well as in the posterior funiculus, where the lesions were present only ipsilateral to the injection side. We found no differences in anti-TMEV lymphoproliferative responses between groups. This suggests that axonal injury itself could contribute to the recruitment of inflammatory cells into the CNS by altering the local microenvironment, such as activating microglia. Recent studies showed that neural precursor cells (NPCs) attenuate experimental autoimmune encephalomyelitis (EAE) after intracerebroventricular or intravenous (IV) injection. NPC therapy in EAE was associated with a decrease in brain inflammation and in tissue injury. Here we studied the mechanism by which IV injected NPCs inhibit myelin oligodendrocyte glycoprotein (MOG)-induced EAE in C57BL/6 mice. IV NPC therapy at 8 days post EAE induction attenuated the clinical course of EAE, and reduced brain inflammation and tissue injury. Microscopical analysis revealed that NPCs did not enter the brain, but were detected in lymph nodes and spleen. The direct interactions between NPCs and lymph node cells (LNC) were examined in vitro. Co-culture experiments showed that neurospheres attenuated significantly the proliferation of EAE-derived T-cells in response to MOG, as well as of naïve T-cells, in response to the non-specific activator ConA. Neurospheres also inhibited MOG-induced production of proinflammatory cytokines in LNCs. Analysis of CFSE emission descent, IL2Rα expression, DNA content and Annexin-V expression indicated that NPCs block T-cell activation and proliferation, rather than induce LNC death. Finally, to examine the relevance of NPC-LNC interactions in vivo, we utilized a proteolipid (PLP)-induced transfer EAE model in SJL/J mice. Transfer of LNCs derived from NPC-treated, PLP-immunized mice into naïve recipients caused a significantly milder disease than transfer of LNCs from non-treated mice. This indicated that NPC therapy reduces the encephalitogenicity of LNCs. Our findings suggest a peripheral immunosuppressive effect of IV-injected NPCs, by a non-specific inhibitory effect on activation and proliferation of lymphocytes in the lymph nodes. WS13-02 Neural stem/precursor cell (NPC) transplantation in experimental autoimmune encephalomyelitis in mice: Unraveling the molecular bases of NPC-dependent immunomodulation Transplantation of undifferentiated adult neural stem/precursor cells (aNPCs) promotes long-lasting neuroprotection in experimental multiple sclerosis by remyelination of injured central nervous system (CSN) axons as well as by immunomodulatory functions. After systemic transplantation, aNPCs enter brain and spinal cord, selectively reach inflamed CNS areasthe "atypical perivascular niches"where major stem cell regulators are focally (re)expressedand survive in vivo for up to 120 days after transplantation. To further explore the immunomodulatory properties of adult neural precursor cells (aNPCs) in mice with experimental autoimmune encephalomyelitis (EAE), we have intravenously (i.v. SJL female mice were immunized subcutaneously with proteolipid protein (PLP)139-151 and i.v. Transplanted aNPCs accumulated and persisted over 100 days after transplantation within major secondary lymphoid organs, whereagainmajor stem cell regulators (e.g., FGF-II, Notch I, BMPs, etc.) were dynamically (e.g., after 15, 30 and 100 days after the immunization) expressed at protein and mRNA levels. In vitro experiments showed aNPCs efficiently inhibiting antigen-specific proliferation of cocultured PLP-reactive encephalitogenic T cells. Our preliminary data show that systemically-injected aNPCs may survive in vivo even for long periods of time and protect from chronic CNS inflammation owing to their capability to exert immunomodulatory functions within both canonical central (e.g. the CNS) as well as non-canonical peripheral (e.g., lymph nodes) site(s) of accumulation, where stem cell surviving factors are ectopically (re)expressed in response to inflammation. WS13-03 Mesenchymal stem cells treat relapsing-remitting experimental autoimmune encephalomyelitis through a dual effect on inflammation and tissue damage Gerdoni E 1 , Gallo B 2 , Casazza S 1 , Pedemonte E 1 , Musio S 2 , Mancardi GL 1 , Pedotti R 2 , Uccelli A 1 Mesenchymal stem cells (MSCs), a subset of adult stem cells derived from the bone marrow stroma, represent an appealing tool for regenerative medicine. Recent studies have demonstrated that MSCs modulate immune responses supporting their utilization for the treatment of autoimmunity. Thus, we sought verifying whether MSC can cure mice affected by experimental autoimmune encephalomyelitis induced with the peptide 139-151 of the proteolipid protein (PLP), a myelin antigen capable of inducing a potent pathogenic T and B cells response. MSC treated mice showed a significantly milder disease and fewer relapses compared to controls. In vivo PLP specific antibodies titres and antigen-specific T cell response were significantly lower in MSCs treated mice. The adoptive transfer of encephalitogenic PLP sensitized, preconditioned with MSC, induced a milder disease compared to controls. MSC treated encephalitogenic cells showed decreased production of IFNγ and TNFα and did not proliferate upon antigen recall thus were considered anergic. injection, Green Fluorescent Protein (GFP) labeled MSCs were detected inside lymph nodes early upon injection and inside the inflamed CNS at later stage. MSC injection resulted in a decreased number of inflammatory infiltrates, reduced demyelination and apoptotic neural cells and, as consequence, sparing of axons and oligodendrocytes. In contrast, no evidence of GFP labeled neural cells were detected, thus not supporting the hypothesis of trans-differentiation. Overall, we propose that MSC may have a dual function that may be effective for the treatment of MS, an autoimmune disease of the CNS where degeneration of neural cells follows inflammation. Bone marrow (BM) derived mesenchymal stromal stem cells (MSCs) can differentiate under certain circumstances into cells from various tissues. The aim of our study was to investigate the ability of BM-MSCs to migrate into CNS inflamed tissue and differentiate into cells of the neural-glial lineage, in the chronic-EAE model, and to evaluate whether these cells can exert a beneficial clinical effect.MSCs were obtained from GFP-transgenic mice. Cells were cultured with a combination of growth factors (FGF-2/BDNF/FGF-8) and checked for their differentiation properties. MSCs were injected intraventricularly (icv) in C57Bl mice with chronic-EAE at the initial phase of the disease. All animals were followed up clinically for at least two months.In vitro, MSCs showed the ability to differentiate into cells with neural and glial-like morphology and were stained with GFAP+ (∼ 30%), O4+ (∼ 10%), Nestin+ (∼ 10%) and NF160+ (∼20%). Cultured MSCs showed immunomodulatory properties and significantly downregulated lymphocyte proliferations against myelin antigens.In vivo, icv injection of MSCs suppressed the clinical (mortality 0%, vs. 16% in the controls; mean maximal score: 3.08, vs. 3.85) and histopathological manifestations of chronic-EAE and induced significant neuroprotection (>90% of axons unaffected in the treated animals vs. 40% in the controls). MSCs migrated into the inflamed lesions and were found to present immunohistological characteristics of neuronal and glial cells.Our results indicate that autologous BM can provide a source of stem cells which downregulate the clinicopathological signs of chronic-EAE and carry a neuroprotective potential; such treatment protocols may be applicable in diseases like multiple sclerosis. WS13-05 Injection of human neurospheres ameliorates a non-human primate model of multiple sclerosis We have transplanted human foetal NPCs in non-human primates with experimental autoimmune encephalomyelitis (EAE). In order to detect also sub clinical EAE, immunized monkeys underwent serial (every 3 weeks after the immunization) brain magnetic resonance (MR). At both clinical or subclinical disease onset, marmosets were injected either intravenously (i.v., n = 5) or intrathecally (i.e., into the cerebrospinal fluid [CSF], n = 4). The remaining six monkeys were used as sham-treated controls. NPCs had been in vitro prelabeled by inserting in them the green fluorescent protein (GFP) gene through 2nd generation lentiviral-base infection. Clinical score and weight were daily monitored along a 90 day-long follow up period. EAE monkeys transplanted i.v. of relapses per day of follow-up; (iii) no. Reduction of demyelination and axonal loss were observed in all transplanted marmosets. At histopathology the great majority of transplanted cells accumulated within the chronicallyinflamed CNS and persisted over time around inflamed CNS perivascular areas without displaying major features of terminal differentiation. These results are of critical and propedeutical importance to envisage the future use of hFNPCs as a safe and efficaciously neuroprotective therapy in MS. WS13-06 Cellular and molecular analysis of SVZ-resident neural stem cells in mice affected by chronic experimental autoimmune encephalomyelitis (EAE) ⁎ These authors contributed equally to the work.We have adopted a multidisciplinary approach to analyse kynetics of sub-ventricular zone (SVZ)-resident neural stem/precursor cells (NPCs) during chronic experimental autoimmune encephalomyelitis (EAE) in mice. Adult C57Bl/6 female mice were immunized with myelinoligodendrocyte glycoprotein (MOG)35-55 and combination of in vivo, in situ and ex vivo analyses performed at different timepoints after the immunization. Fast-and slow-cycling SVZ-resident NPCs were studied by intraperitoneal injection with pulsed IddU/BrdU prior to sacrification. The orientation of mitotic cleavage of endogenous dividing NPCs (namely a measure of the (sub)fraction of NPCs undergoing asymmetric vs. symmetric cell division), was also studied. Early (e.g., 20 days after the immunization) increase of BrdU incorporation by both SVZ-resident glial and neuronal progenitors was revealed at confocal microscopy. Parallel in vitro data showed progressive impairment of clonal efficiency within neurosphere cell lines established from EAE mice, starting at 20 dpi, peaking at 30 dpi and then slowly but insufficiently recovering at later time points. No differences of sphere size and in vitro growth rate were found between groups.Our preliminary results show increased cell proliferation within the SVZ neurogenic niche paralleled by striking quantitative impairment of the endogenous neural stem cell compartment. Further studies aiming at the identification of cellular and molecular pathways involved in reactivation/reprogramming of the cell type(s) triggered by the lesion(s) may help solve and/or improve the contribution of endogenous NPCs to repair mechanisms. MS Centre ErasMS, Rotterdam, The Netherlands Adenosine is released at sites of inflammation and tissue damage and activates adenosine receptors. Many of the reported adenosine receptormediated effects are neuroprotective. However, adenosine may also aggravate neuronal injury by promoting inflammation.We have recently demonstrated an anti-inflammatory function for foamy macrophages in multiple sclerosis (MS) lesions and hypothesized that expression of adenosine receptors is altered in these cells thereby affecting their function. Therefore, we determined mRNA expression levels of the adenosine receptors (A1, A2a, A2b, and A3) in human myelin-laden macrophages in vitro and in MS lesions. A3 receptor mRNA expression was significantly downregulated (5-fold) in foamy macrophages as compared to control macrophages. LPS strongly induced A2a mRNA expression in both control and foamy macrophages (100-fold). In contrast, LPS synergistically downregulated A3 receptor mRNA (100-fold) in foamy macrophages compared to control macrophages. To assess whether this potent mRNA regulation also occurs during MS, expression levels were determined in MS brain and brain tissue of non-demented controls. A3 receptor mRNA was significantly upregulated in MS brain and the expression correlated with lesion activity. Immunohistochemistry will reveal which cells are expressing A3 receptors in MS brain.Our data demonstrate that adenosine receptor expression in MS brain and on foamy macrophages in vitro is strongly regulated. The altered balance of the expression levels of the different adenosine receptors is likely to influence the functional response to adenosine. Unraveling the function of these immune-modulatory receptors will allow better understanding of endogenous neuroprotective mechanisms and may thereby open new roads to disease intervention.WS14-02 NR4A2 (Nurr1), an orphan nuclear receptor, is overexpressed in peripheral blood T lymphocytes of multiple sclerosis Yoshimitsu Doi, Shinji Oki, Jun-ichi Satoh, Toshimasa Aranami, Sachiko Miyake, Takashi Yamamura Department of Immunology, National Institute of Neuroscience, NCNP, Tokyo, JapanObjective: To study a role of the NR4A subfamily of nuclear receptors in the immunopathogenesis of multiple sclerosis (MS). Background: Nurrelated factor-1 (Nurr1; NR4A2), nerve growth factor-induced gene-B (NGFI-B; NR4A1), and neuron-related orphan receptor-1 (NOR-1; NR4A3) are transcription factors of the steroid/thyroid receptor superfamily, rapidly induced by exposure to growth factors and cytokines, suggesting a biological role of cell growth, differentiation and apoptosis (Pei et al. By analyzing a cDNA microarray, we found that NR4A2 is upregulated in CD3 + T cells of MS patients compared with healthy control (CN) subjects (Satoh et al. However, an active role of NR4A2 and its transcriptional targets in MS remain unknown. Methods: NR4A1, NR4A2, NR4A3, and OPN mRNA levels were quantitatively analyzed in cDNA of CD3 + T cells of 58 untreated active MS patients and 19 CN subjects by real-time PCR.Results: NR4A2 and NR4A3 were significantly upregulated in MS T cells, but NR4A2 levels did not correlate with those of OPN. Conclusions: Quantitative real-time RT-PCR confirms upregulation of NR4A2 in MS T cells. Although specific transcriptional targets of NR4A2 in T cells remain unknown, NR4A2 might play a role in immunopathogenetic mechanisms of MS patients. WS14-03 Genomic evidence for subtypes of Multiple Sclerosis: Presence of a viral response signature in a subpopulation of patients Despite epidemiological support for this hypothesis, the presence and physiological significance of viral infections in MS is still unclear. Comparative analysis identified a stereotyped gene expression program that appears to reflect a virus response program, in the peripheral blood cells of patients with RRMS. Subsequent analysis revealed that the virus response program was characteristic of not all, but approximately half of the RRMS patients. Thus, the transcriptional signature of the peripheral blood cells defines a subpopulation of RRMS patients who exhibit evidence for a link between viruses and MS. The mechanisms responsible for the localization of lesions in multiple sclerosis (MS) are currently unknown. To investigate this, we performed HLA-DR and HLA-DQ typing and examined T-cell reactivity to myelin proteins in 121 patients with MS not on immunomodulatory therapy, 71 healthy subjects and 47 patients with other diseases of the central nervous system (CNS). We found that 47.0% of MS patients with brainstem or cerebellar lesions, as determined by clinical assessment and/or magnetic resonance imaging, had increased circulating T-cell reactivity to an immunodominant region of myelin proteolipid protein ), compared to 10.0% of MS patients without brainstem or cerebellar lesions, 11.3% of healthy subjects and 19.1% of patients with other CNS diseases (p < 0.0006). We also found that 78.6% of MS patients with brainstem or cerebellar lesions carried HLA-DR4, DR7 or DR13 alleles, whereas only 28.9% of patients without brainstem or cerebellar lesions carried these alleles (p = 0.00000003). Furthermore, 60.9% of MS patients carrying HLA-DR4, DR7 or DR13 alleles had increased T-cell reactivity to PLP 184-209 , compared to 12.2% of MS patients not carrying these HLA alleles (p = 0.0001). Interestingly, H-2 k mice immunized with PLP 184-209 also develop inflammatory demyelinating lesions predominantly in the brainstem and cerebellum. Our results indicate that T-cell reactivity to PLP 184-209 drives the development of brainstem and cerebellar lesions in MS. In patients carrying HLA-DR4, DR7 or DR13 alleles, therapy with altered peptide ligands based on PLP 184-209 might prevent the development of brainstem and cerebellar lesions. WS14-05 A system biology approach to the immune system network reveals functional differences in health and Multiple Sclerosis P. Villoslada, R. Palacios, J. Goñi, N. Velez, J. Sepulcre Neuroimmunology lab, Department of Neurology, University of Navarra, Spain Background: A functional knowledge of the properties of the gene networks that control biological functions might be critical for understanding complex diseases, such as Multiple Sclerosis (MS). Our aim was to model the gene network that controls the T-cell activation-differentiation-suppression process, which is critical for the development of MS, and to assess its functional properties in health and autoimmunity. Methods: We modelled a network with 20 critical immune system genes using scientific text mining and experimental data from 52 healthy controls and 52 patients with MS obtained by real-time PCR by using a Bayesian approach. We compared the functional differences in the network in health and autoimmunity using the Kullback-Leibler (KL) divergence for each pair of nodes. Results: We found a network with 31 links (gene-interactions), 18 already known (expected) and 13 non previously described. We found significant differences between the control and MS network, mainly in the regulatory (TGFB1, IL10, JAG1) and proinflammatory pathways (IL-12). In addition we identified the effect of interferon beta therapy in the network, mainly in the interactions between ITGA4-TGFB1 and JAG1-TNF, TGFB1-GATA3, IL10-CD28, TGFB1-ITGB7, ITGA4-ITGB7 and IL10-ITGA4. Conclusion: Our results indicate that assessing the properties of the immune gene network using a system biology approach reveals significant changes in autoimmunity and provides insights in the effect of immunomodulatory therapy. Objective: To characterize T-cell infiltrates and in vivo expanded clonotypes derived from MS lesions. Background: Oligoclonally expanded T-lymphocytes perpetuate inflammatory response against CNS myelin antigens. Methods: Brain and spinal cord tissue was obtained six hours post mortem from a female patient with aggressive relapsing remitting (RR) MS. Lesions were characterized based on the longitudinal CNS MRI, the last one 4 days prior to death, and histopathological and immunohistochemistry studies. Viable cells from six lesions and normal appearing white matter (NAWM) were expanded in vitro. To study in vivo expanded clonotypes and their TCRVb usage in the infiltrates from the lesions and NAWM, we RT-PCRamplified TCR genes and performed single-strand conformational polymorphism (SSCP) pattern analysis. Results: Antigen specificity of MS-lesion derived T-cells was tested against a panel of immunodominant myelin-derived peptides. The highest proliferative response was detected against PLP 190-209 in acute pontine, chronic periventricular and thoracic spine lesions. Cells derived from acute lesion exhibited proliferative response against six myelin peptides, four of which were recognized in chronic thoracic, and three in chronic periventricular lesion. Single-strand conformational polymorphism (SSCP) pattern analysis identified limited number of TCRVB chains expressed correlating to the in vivo expanded clonotypes. It revealed distinct patterns of TCR VB family chains in each MS lesion and NAWM. However, TCRVB4, VB5.1, VB8, and VB13.1 were present in all lesions. Antigen specificity of the clones corresponding to the in vivo expanded clonotypes is presently characterized using synthetic combinatorial peptide libraries. Relevance: This is the first study to characterize post-mortem derived T-cell infiltrates from MS lesions at various stages of development. OS1A-01 Endothelial cell Weibel-Palade bodies regulate blood brain barrier function and susceptibility to experimental allergic encephalomyelitis Pertussis toxin (PTX), an immunopotentiating adjuvant in experimental allergic encephalomyelitis (EAE), also elicits leukocytosis, alters glucose regulation, vascular permeability and blood-tissue barrier functions, and sensitizes the vascular endothelium to vasoactive amines such as histamine (BPHS). The mechanism underlying BPHS is unknown but requires a 2-3 day latency and lasts ∼ 30 days. This suggests that the induction phase is associated with the storage of preformed vasoactive factors that are then released during the effector phase. Weibel-Palade bodies (WPB) within endothelial cells (EC) are secretory granules known to release preformed chemokines, adhesion molecules and von Willebrand Factor (Vwf) following exposure to histamine. Mice with a disrupted Vwf allele (VwfKO) have EC that are deficient in WPB and were used to evaluate the role of this pathway in BPHS and susceptibility to EAE. No significant differences in susceptibility to BPHS between wild-type and VwfKO mice were detected at three days; however, in VwfKO mice sensitivity persisted longer and the LD 50 -histamine was lower at later time points. Correspondingly, the onset of EAE was earlier, disease more severe and blood brain barrier (BBB) permeability significantly increased in VwfKO mice compared to wild-type mice. Increased BBB permeability in VwfKO mice was not due to increased encephalitogenic T-cell activity since BBB permeability was significantly greater in PTX-treated VwfKO mice compared to mice receiving encephalitogen-complete Freund's adjuvant (CFA) + PTX, CFA + PTX or CFA. Taken together, these data indicate that WPB normally repress BPHS and adjuvant-induced alterations in BBB function associated with actively induced EAE. OS1A-02 Comparison of monocyte and T lymphocyte trafficking at the blood retinal barrier in vivo H. Xu a , A. Manivannan b , R. Dawson a ; I.J. Crane a , and J. Liversidge a a Department of Ophthalmology, b Department of Radiology, University of Aberdeen, Scotland, UK Neural inflammation is caused by lymphocyte and monocyte infiltration across the tight junctions of blood-brain barrier, but the mechanisms involved in the adhesion and transendothelial migration of different leukocyte subsets are unclear. Using scanning laser ophthalmoscopy we have investigated the trafficking of monocytes and compared this with Th1 and Th2 cell trafficking at the blood-retina barrier (BRB) of the neuroretina, an extended organ of the brain. In normal circumstances, retinal vessels are subjected to high shear stresses and leukocytes do not roll. Activated T cells can migrate into the neuroretina in the absence of rolling and adhesion molecule LFA-1 is critical for the transendothelial migration. In inflamed retina there is a reduced shear stress in both arterioles and venules at the BRB but leukocytes only roll in the venules. Naïve T cells and monocytes roll at similar levels, however primed/activated T cells roll in a higher efficiency. Different adhesion molecules are responsible for the rolling of different leukocyte subsets. In vivo conditioned monocytes roll faster than primed/activated T cells, and this fast rolling appears to be L-selectin dependent. PSGL-1 is also involved in the rolling of monocytes and Th1 but not Th2 cells, whereas CD44 is involved in the rolling of all activated leukocytes. For transendothelial migration, LFA-1 is crucial for Th1 and Th2 cell diapedesis but not monocytes. In conclusion, reduced shear stress is required for leukocyte to roll at the BRB. Different adhesion molecules are involved in the trafficking of different leukocyte subsets at the BRB. Thrombospondin-1 (TSP-1) serves as a ligand for several receptors present on the surface of immune cells, including VLA-4. Because TSP-1 is expressed at high levels by endothelial cells, we investigated its role in the adhesion and traffic of inflammatory cells in experimental allergic encephalomyelitis (EAE). Using in vitro techniques for the study of adhesion and migration under blood flow conditions we show that by virtue of its ability to bind VLA-4, TSP-1 is a potent substrate for the adhesion of Th1 cells and macrophages. This function appears to be critical in vivo, as TSP-1 null mice (C57BL/6 background) are completely resistant to developing MOG-induced EAE, in spite of their ability to generate normal immune responses against MOG [i.e., normal proliferation, antibody and cytokine (IL-2, IFN-γ, IL-4, IL-10, IL-17 and TGF-β), production upon rechallenge with MOG in vitro]. An in vivo role in trafficking for TSP-1 was also suggested by our findings using immunohistochemistry that TSP-1 is expressed constitutively at high levels by perimeningeal blood vessels in the CNS. This was further supported by histological and flow cytometric analyses performed at various intervals post-EAE induction, which showed virtually a complete absence of macrophages and other inflammatory cell infiltrates in the CNS of TSP-1 null mice. Thus our findings suggest that TSP-1 serves as an adhesion molecule, and that its expression by blood vessels is required for the traffic of inflammatory cells into the CNS parenchyma. OS1A-04 Small G protein RhoA controls lymphocyte recruitment in brain vessels and the induction of experimental autoimmune encephalomyelitis Department of Pathology, University of Verona, Verona, Italy We have recently demonstrated that 23-40 domain of small G protein RhoA controls integrin affinity, while 92-119 RhoA domain controls lateral mobility and clustering in naïve T cells, showing that RhoA controls both modalities of integrin activation. The goal of this study was to determine the role of RhoA in the recruitment of autoreactive T lymphocytes in inflamed brain vessels and in the induction of experimental autoimmune encephalomyelitis (EAE).Methods: We used 3 trojan peptides able to block RhoA effector regions, consisting of a sequence of Antennapedia (P1) able to translocate through the cell membranes linked to: (1) 23-40; or (2) 92-119; or (3) 75-92 domains of RhoA. Intravital microscopy in inflamed mouse brain microcirculation and induction of EAE by adoptive transfer of PLP139-151 reactive T cells were performed.Results: All P1-derived Trojan peptides did not alter intracellular calcium release, adhesion molecules expression and distribution, and proliferation of PLP139-151 specific T cells. Intravital microscopy studies showed that P1/ 23-40 and P1/92-119 trojan peptides inhibit stable adhesion of encephalitogenic T lymphocytes in inflamed brain venules without affecting rolling, while, in contrast, controls P1 and P1/75-92 peptides were ineffective. Moreover RhoA peptide P1-92-119, but not P1-23-40, was able to block transfer EAE, induced by PLP139-151 specific CD4 + T cells: the incidence of the disease and the mean clinical score were significantly lower in animals treated with RhoA peptide P1-92-119. Conclusion: Selective regions of RhoA control autoreactive T cells migration into the inflamed brain and have a critical role in the induction of EAE. OS1A-05 Signaling through MyD88 regulates leukocyte recruitment after brain injury Injury to the central nervous system (CNS) provokes an innate inflammatory reaction that engages infiltrating leukocytes with the capacity to repair and/or exacerbate tissue damage. The initial cues that orchestrate leukocyte entry to the injured CNS remain poorly-defined. We have used flow cytometry to investigate whether MyD88, an adaptor protein that transmits signals from Toll-like receptors (TLRs) and receptors for interleukin (IL)-1 and IL-18, regulates leukocyte infiltration to the stabinjured entorhinal cortex (EC) and to sites of axonal degeneration in the denervated hippocampus. We have previously established the kinetics of leukocyte entry to the denervated hippocampus. Whereas T cells showed small, gradual increases over 5 days, macrophage infiltration was pronounced and peaked within 12-24 h. Infiltration coincided with increased mRNA for matrix-metalloproteinase-12 and a disintegrin and metalloproteinase (ADAM)-12, but not interferon-γ or IL-17. MyD88-deficiency reduced macrophage infiltration to the stab-injured EC and the denervated hippocampus by more than 75% at 5 days postinjury. MyD88-deficiency abrogated T cell infiltration to the denervated hippocampus at this time, and reduced T cell entry to the stab-injured EC by 50%. MyD88-deficiency strongly reduced stab-injury induced transcripts for TNFα and CCL2, which were increased several hundred fold at time of peak expression in wildtype mice (3 h) . Stab injury-induced leukocyte recruitment and gene expression were unaffected by TLR2 or TLR4 deficiency. These data show that MyD88-dependent signaling is important for proinflammatory gene expression and leukocyte recruitment after CNS injury. The chemokine receptor 2 (CCR2) is thought to play an important role in the induction of autoimmune disease in the central nervous system (CNS) by attracting immune effector cells from the periphery into the brain. Oral The aim of this study is to develop a technique that would lead to a loss of function of CCR2 in hematopoietic cells in order to reduce the infiltration of blood borne cells into the brain in experimental autoimmune encephalomyelitis (EAE). Using bone marrow (BM) chimeric mice, we demonstrate that expression of CCR2 on hematopoietic cells, but not on radioresistant CNS-resident cells, determines EAE disease course. Moreover, we find that mixed chimeras with BM from RAG1 −/− × CCR2 −/− mice are highly susceptible to disease, suggesting that CCR2 expression on T and B lymphocytes is not required for EAE development. We next generated a mutant murine CCR2 (CCR2Y152F) by site-directed mutagenesis. When HEK293 cells were transfected with wildtype CCR2 and stimulated with the CCR2 ligand, monocyte chemoattractant protein-1, a rapid and transient increase in intracellular calcium concentration was observed. This calcium response was completely abolished by cotransfection of HEK293 cells with CCR2Y152F and wildtype CCR2. Transduction of adult hematopoietic stem cells with CCR2Y152F and subsequent generation of BM chimeric mice will be used to determine the contribution of CCR2 on myeloid cells to the inflammatory response in EAE. OS1B-02 Interferon-gamma induced chemokines and pertussis toxin synergize to promote T cell entry to the central nervous system Inflammation of the central nervous system (CNS), which occurs during multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), is characterized by increased levels of interferon-gamma (IFNγ), a cytokine not normally expressed in the CNS. This resulted in stable, long-lived expression of ELISA-detectable IFNγ in cerebrospinal fluid. Other chemokines (CXCL2, CCL2, CCL3) were not induced. Mice lacking the IFNγ receptor showed no response, and a control viral vector did not induce chemokine expression. Chemokine expression was predominantly localized to meningeal and ependymal cells, and was also seen in astrocytes and microglia. However, when pertussis toxin was given intraperitoneally to mice infected with the IFNγ vector, there was a dramatic increase in the number of T lymphocytes detected in the CNS by flow cytometry. This increase in blood-derived immune cells in the CNS did not occur with pertussis toxin alone, and did not manifest as histologically detectable inflammatory pathology. These results show that IFNγ induces a characteristic glial chemokine response that by itself is insufficient to promote inflammation, but that CNS chemoattractant signals can synergize with a peripheral infectious stimulus to drive T cell entry into the CNS. OS1B-03 Antigen presentation capacity by glial cell types during CNS infection Acute CNS infection by a nonlethal neurotropic coronavirus is controlled by CD8 T lymphocytes. Perforin mediated cytolysis controls virus replication in macrophages/microglia and astrocytes, implicating TcR-class I interactions. By contrast, IFN-gamma controls virus replication in oligodendroglia, suggesting an inherent resistance to cytolysis. Despite these effector functions and T cell retention, virus persists in glia resulting in ongoing demyelination. To investigate the molecular basis of glia-T cell interactions in vivo, microglia, astrocytes and oligodendroglia were isolated from infected mice and analyzed for MHC expression and mRNA encoding antigen processing molecules. Enhanced class I expression was consistent with earlier transcription of genes associated with the antigenprocessing machinery, specifically TAP1 and LMP2, in microglia compared to oligodendroglia. Furthermore, the biphasic transcription profile observed in microglia contrasted with the sharp expression peak in oligodendroglia, coinciding with maximal T cell effector function. Oligodendroglia were also distinct from microglia by their strong upregulation of the CD8 T cell inhibitory ligand PDL-1, substantiating strict control of T cell effector function. Contrasting class I expression, class II upregulation was extensive on microglia, but very limited on oligodendroglia and astrocytes. In summary, microglia have enhanced and earlier potential to present antigen compared to oligodendroglia and astrocytes, due to distinct responses to Type I-and Type II-IFN. Nevertheless, prolonged antigen presentation capacity implicated by sustained MHC expression in all glial cell types may be muted by differential expression of modulatory molecules. OS1B-04 Immune-mediated loss of neurons secondary to the destruction of supporting astrocytes Neuronal injury and loss is an increasingly recognized component of the pathology of multiple sclerosis (MS). Published data indicate that T cells can mediate neuronal damage in an MHC/antigen-restricted as well as an MHC-unrestricted manner, while sparing astrocytes. In contrast, glia-directed neuro-cytotoxicity has been shown to occur in vitro by innate immune-cells such as γ/δ T cells and natural killer (NK) cells. Here, we sought to determine if NK cells could damage human neurons. To do so, we utilized CD56 + NK cells from IL-2-activated, normal adult blood, and primary human neuron-glia cultures derived from fetal brain tissue. Interestingly, we found that although co-incubation of NK cells with neuron-glia cultures resulted in detachment of neurons, the neuron viability as determined by propidium iodide exclusion was normal at all time points examined. In contrast, the underlying astrocytes, which constitute up to 10% of these cultures, were very efficiently killed by the NK cells. Time-lapse video microscopy indicated that NK cells began targeting the glial cells within minutes after co-incubation, whereas neurons were largely ignored. After 24 h, the remaining neurons were found to clump on the few surviving glial cells as determined by confocal microscopy. This data is consistent with a model of indirect neuronal injury mediated by innate immune cells in which neuron loss reflects primary loss of glial, trophic, or substrate support. OS1B-05 Invariant Vα19-Jα33 NKT cells promote ICOS-dependent IL-10 production by B cells which regulate inflammation in a model of multiple sclerosis J. Ludovic Croxford a , Sachiko Miyake a , Michio Shimamura b , and Takashi Yamamura a a Department of Immunology, National Institute of Neuroscience, Tokyo Japan; b Developmental Immunology Unit, Mitsubishi Kagaku Institute, Tokyo, JapanRecently, a new population of CD1-independent NKT cells, restricted by MR1, a non-classical MHC class I molecule, have been described. MR1restricted NKT cells express the invariant Vα19-Jα33 T cell receptor (Vα19 + NKT). However, their function in immunity is unknown. In humans, Vα7.2-Jα33 NKT cells (homologous to Vα19 + NKT cells) were detected in multiple sclerosis (MS) inflammatory CNS lesions. Therefore, we determined the role of Vα19-Jα33 NKT cells in experimental autoimmune encephalomyelitis (EAE), a T-cell mediated autoimmune demyelinating disease of the CNS and model of MS. In contrast to humans, mice have extremely low numbers of Vα19 NKT cells. Therefore we used Vα19 TCR Tg mice crossed to CD1 deficient (CD1KO) mice which lack Vα14 NKT cells. We observed that over-expression of Vα19 NKT cells protects mice from EAE, and is associated with a decrease of Th1 cytokines but an increase in IL-10. In addition, adoptive transfer of Vα19 NKT cells to wt EAE mice could also inhibit clinical disease. In contrast, mice deficient in Vα19 NKT cells (MR1KO) exhibited exacerbated EAE. Coculture of Vα19 NKT cells with B cells demonstrated that B cells are induced to produce IL-10, an anti-inflammatory cytokine known to inhibit EAE. IL-10 production from B cells was ICOS-dependent but MR1 independent. This study demonstrates that Vα19 NKT cells have a regulatory function in EAE and therefore may be attractive therapeutic targets for the therapy of MS and other autoimmune diseases. Activation of Vα19 NKT cells by novel glycolipids may allow for disease-specific therapy.Oral Session 2A: Immune-mediated pathology and neuroprotection (1) 86 Abstracts OS2A-01 Immunomodulation of microglial phenotypes: Implications for multiple sclerosis and Alzheimer's disease Cell renewal in the adult central nervous system (CNS) is limited, and is blocked under inflammatory brain conditions. Microglia play a major role in the cellular immune response associated with neurodegeneration. We show that T-cell-derived cytokines (IFN-γ and IL-4) can induce microglia to become neuroprotective and to induce cell renewal. In contrast, aggregated β-amyloid (Aβ) induces microglia to become cytotoxic and block both neurogenesis and oligodendrogenesis of adult rodent neural stem/progenitor cells (NPCs) In addition, high levels but not low levels of interferon-γ (IFNγ, a cytokine associated with inflammatory autoimmune diseases, confer on rodent microglia a phenotype that impedes oligodendrogenesis from NPCs. IL-4, reversed the impediment, attenuated TNF-α production, and overcame blockage of insulin like growth factor (IGF)-I production.. In rodents with acute or chronic experimental autoimmune encephalomyelitis, injection of IL-4-activated microglia into the cerebral spinal fluid resulted in improved clinical symptoms and increased oligodendrogenesis from the endogenous stem cell pool in the spinal cord, and induced neurogenesis in dentate gyrus spatially associated with microglia expressing MHC-II and IGF-I. Using double-transgenic mice expressing mutant human genes encoding presenilin-1 and amyloid precursor protein (a mouse model of Alzheimer's disease), we show that modulation of microglia into dendritic-like cells, achieved by a T cell-based vaccination with copolymer-1, resulted in reduction of cognitive decline, elimination of plaque formation, and induction of neuronal survival and neurogenesis. These results introduce a new microglia phenotype as necessary players in fighting off neurodegenerative conditions. OS2A-02 β-Amyloid uptake by microglial cells and proinflammatory cytokines S. Kataoka, H. Hara and T. Tabira National Institute for Longevity Sciences, Obu, Japan Alzheimer disease (AD) is characterized by extracellular deposits of fibrillar β-amyloid (Aβ) in the brain, accompanied with abundant phenotypically activated microglia in the brain. Recent studies showed that anti-Aβ immunotherapy reduced amyloid burden in the brains of AD model mice and that Aβ-specific antibodies enhanced Aβ phagocytosis by microglial cells in an IgG receptor (FcR) dependent manner. We addressed whether or not proinflammatory cytokines could influence anti-Aβ antibody-mediated phagocytosis of fAβ by microglia. Using aggregated Aβ and fluorescent-labeled lysosomal probes, we examined phagocytosis of fibrillar Aβ (fAβ) and lysosomal pathway in mouse primary cultured microglial cells and murine Ra2 microglial cell line in the presence or in the absence of anti-Aβ antibodies and proinflammatory cytokines. Anti-Aβ antibodies drastically enhanced fAβ phagocytosis, which anti-CD16/CD32 antibody blocked by 70%. Proinflammatory cytokines such as interleukine-6 (IL-6), tumor necrosis factor α (TNFα) and interferon-γ (IFN-γ) decreased Aβ uptake as well as lyososomal vesicle formation. Proinflammatory cytokines did not inhibit Aβ uptake induced by anti-Aβ antibody. However, anti-Aβ antibody treatment in the presence of proinflammatory cytokines caused microglial cell damages characterized by large cytoplasmic vacuoles. Our results show that Aβ-specific antibodies release the proinflammatory cytokine-induced suppression of Aβ phagocytosis by microglial cells, but that Aβ phagocytosis under proinflammatory condition leads to microglial dysfunction. These findings support Aβ vaccination-based therapy combined with anti-inflammatory therapy for clearing Aβ deposits in the AD brain. OS2A-03 New mechanism underlying glatiramer acetate mediated neuroprotection Jianuo Liu, Jamie Lin, Peter Olds, Jason G. Glanzer, Tatiana K. Bronich, Steve Caplan, Yuri Persidsky, Howard E. Gendelman and Jonathan Kipnis University of Nebraska Medical Center, Omaha, USA Glatiramer Acetate (GA) is a synthetic copolymer and has been shown to induce neuroprotection under several acute and chronic neurodegenerative conditions. The mechanism underlying its neuroprotective activities, however, remains obscure. Many studies have focused on the role of T cells induced by GA administration, and particularly on the GA-induced Th2 shift in the profile of the induced T cell response. Since GA can induce rapid neuroprotection following brain trauma or glutamate-intoxication of retinal ganglion cells on a time scale that is insufficient for a T cell mediated response, we hypothesized that GA-induced neuroprotection might be T cell-independent and that a direct effect of GA on neurons might exist. This direct neuroprotective effect of GA is associated with its internalization into primary neuronal cells and increase expression of BDNF and activation of PKCα. Moreover, GA enters mononuclear phagocytes via induction of the RhoA pathway and causes an alteration of actin organization, which increases the trans-migratory abilities of monocytes and dendritic cells (DCs) through the blood brain barrier (BBB). DCs loaded with GA (DC vaccination) attenuate degeneration of injured optic nerve fibers as observed in our in vivo optic nerve crush injury model. This novel mechanism of GA-mediated neuroprotection may lead to development of new therapeutic regimens for various neurodegenerative as well as autoimmune diseases. OS2A-04 IL-10-producing CD4 + CD25 + T cells play an important role in reduction of neuronal death in a stroke model Inflammation plays an important role in ischemic stroke and in humans IL-10 may have a beneficial effect in stroke. We previously demonstrated reduction of infarct size in a mouse stroke model following transfer of CD4 + T-cells from mice tolerized with myelin oligodendrocyte glycoprotein (MOG) (35-55); CD4 + T cells from nasally tolerized IL-10-deficient mice had no effect. We found less neuronal death and axonal damage as shown by MAP2 staining and SMI-32 staining following nasal tolerization with MOG in wt animals compared to IL-10 −/− mice. The cytokine profile of splenocytes from nasally-tolerized IL-10 −/− mice revealed a reduction in IFN-γ compared to non tolerized littermates and was associated with increased TGF-beta production and elevation in the frequency of LAP + CD4 + T-cells. These results demonstrate the lack of neuroprotection observed in IL-10 −/− mice vs. wt mice relates to the absence of IL-10 rather than a lack of tolerance. Flow cytometry analysis showed elevation in the frequency of CD4 + CD25 + T-cells following nasal treatment in wt but not in IL-10 −/− mice. To further investigate the neuroprotective role of CD4 + CD25 + T-cells, we incubated mouse neuronal cultures with CD25-depleted CD4 + T cells. We found increased neuronal cell death following oxidative stress in depleted cultures. Moreover, incubation of mouse neuronal cultures with CD4 + CD25 + T cells together with anti-IL-10 antibody significantly reduced the neuroprotective effect of CD4 + CD25 + T cells. In conclusion, our data suggest that IL-10-producing CD4 + CD25 + T cells play an important role in reduction of neuronal cell death following brain ischemic injury. OS2A-05 EPO mediated neuroprotection against ischemic and excitotoxic injury requires TNFRI signaling Tumour Necrosis Factor Receptor I (TNFRI) and its ligand TNF are constitutively expressed by CNS neurons and are upregulated after brain injury. TNF has been shown to exert direct neuroprotective effects by maintaining calcium homeostasis, decreasing glutamate-induced currents, and activating a cell protective pathway that involves inhibition of caspase 8, upregulation of FLICE-like inhibitory protein (FLIP L ) and induction of p50/ p65 NF-κB (Taoufik et al., under submission) . The tissue protective cytokine erythropoietin (EPO) and its receptor EPO-R are expressed by CNS neurons and their expression is upregulated following injury. EPO exerts potent neurotrophic properties and can prevent neuronal apoptosis after metabolic stress, cerebral ischemia, traumatic brain injury, spinal cord injury and kainate induced seizures.In this study we show that TNF and TNFRI are necessary for induction of the EPO/EPO-R neuroprotective pathway following ischemic and excitotoxic injury. Using TNFRI deficient mice and in vivo models of ischemic and excitotoxic injury (permanent middle cerebral artery occlusion (MCAO) and kainate induced seizures) we provide evidence that TNFRI signaling is necessary for EPO-mediated neuroprotection. We further show that TNF, acting specifically through TNFRI, induces the expression of EPO and EPOR in primary cortical neurons after glucose deprivation (GD) and oxygen glucose deprivation (OGD) and this is associated with increased NF-κB activation and neuron survival. These results further extend our understanding of the mechanisms of TNF/TNFRI-mediated neuroprotection and provides novel evidence for the beneficial role of brain inflammation in response to injury.Oral Session 2B: Immune-mediated pathology and neuroprotection (2) OS2B-01Immunization with neurofilament light protein induces spastic paresis and axonal degeneration in Biozzi ABH mice Axonal damage is the major cause of irreversible neurological disability in MS patients. While axonal damage correlates with antibodies against neurofilament light (NF-L) protein, a major component of the axonal cytoskeleton, the possible pathogenic role of autoimmunity to axonal antigens such as NF-L has so far been ignored.Here we describe that Biozzi ABH mice immunized with NF-L protein develop neurological disease characterized primarily by spastic paresis concomitant with axonal degeneration and inflammation in the spinal cord and, less so, in the sciatic nerve. In the CNS, F4/80 + macrophages/microglia were abundant while low numbers of CD4 + and CD8 + T-cells were observed. Peripherally, responses of splenocytes to NF-L were characterized by production of the pro-inflammatory cytokines IFN-gamma and TNFalpha. Elevated levels of circulating antibodies recognizing rmNF-L were present in the serum. Immunoglobulin deposits were observed within neuronal cell bodies in the CNS of mice exhibiting clinical disease indicating that antibodies to NF-L may have a pathogenic role in axonal degeneration. We conclude that autoimmunity to NF-L protein induces axonal degeneration and clinical neurological disease in mice. These results indicate that autoimmunity to axonal antigens, as described in MS, may be pathogenic rather than act as merely a surrogate marker for axonal degeneration. OS2B-02 Transgenic inactivation of astroglial NF-κB improves functional recovery following spinal cord injury and experimental autoimmune encephalomyelitis Roberta Brambilla 1 , Kim Esham 1 , Martin Oudega 1 , Andres Hurtado 1 , Scott R. Barnum 2 , John R. Bethea 1In the central nervous system (CNS) the transcription factor NF-κB is a key regulator of inflammation and secondary injury processes. Following trauma or disease, the expression of NF-κB-dependent genes is activated, leading to both protective and detrimental effects on CNS recovery. Here we demonstrate that gene-targeted inactivation of astroglial NF-κB in mice (GFAP-IκBα-dn mice) leads to dramatic improvement in locomotor recovery 8 weeks after spinal cord injury (SCI). Histologically, transgenic mice exhibit increased white matter preservation, with selective sparing of reticulospinal and corticospinal tracts. In parallel, GFAP-IκBα-dn mice show reduced expression of proinflammatory chemokines and cytokines (CXCL10, CCL2, TGFβ2), and of chondroitinsulphate proteoglycans forming the glial scar. Similarly, we demonstrate that GFAP-IκBα-dn mice recover significantly better than wild type (WT) following experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. Transgenic mice develop the demyelinating pathology 5 days later than WT and, chronically, recover locomotor function almost to normality. We conclude that inhibition of NF-κB signaling in astrocytes results in protective effects following CNS injury and propose the NF-κB pathway as a new target for the development of therapeutic strategies to treat such pathologies. OS2B-03 Influence of leukaemia inhibitory factor on macrophage function in vitro and during experimental autoimmune encephalomyelitis Jerome Hendriks, Niels Hellings, Joris Vanderlocht, Piet StinissenHasselt University, Biomedical Research Institute and School of Life Sciences, Transnationale Universiteit Limburg, Diepenbeek, BelgiumIn the chronic disabling disease multiple sclerosis (MS), infiltration of the central nervous system (CNS) by autoreactive T-cells and macrophages results in damage to glial cells and neurons. Recent findings indicate that inflammatory cells are not merely detrimental but may also have protective effects. We previously showed that leukemia inhibitory factor (LIF), a member of the neurokine family of neurotrophic factors, is secreted by T cells and macrophages, and protects oligodendrocytes from TNF-α induced apoptosis. In experimental autoimmune encephalomyelitis (EAE), the animal model for MS, clinical symptoms are suppressed by LIF treatment. We demonstrate that among immune cells mainly macrophages express neurokine receptors. Thus, neurokines may influence macrophage function and thereby act immunomodulatory. Therefore, we investigated whether the neurokines LIF and oncostatin M affect macrophage function in vitro by studying their effect on myelin phagocytosis, pro-inflammatory cytokine production and oxygen radical production, all important processes in MS lesion formation. We are currently evaluating the in vivo effects of neurokines on the inflammatory response during EAE. OS2B-04 Cannabinoid-mediated immunosuppression in experimental allergic encephalomyelitis David Baker 1 , J. Ludovic Croxford 1,2 , Gareth Pryce 1 , Samuel M. Jackson 1 , Catherine Ledent 3 , Giovanni Marsicano 4 , Beat Lutz 4 , Gavin Giovannoni 1 , Roger G. Pertwee 5 and Takashi Yamamura 2 Scientific evidence, initially demonstrated in experimental allergic encephalomyelitis (EAE) models, now supports previously anecdotal evidence that cannabinoids may be beneficial in symptom control in multiple sclerosis. Natural cannabinoids such as tetrahydrocannabinol or synthetic cannabinoid (CB) receptor agonists can also induce immunosuppression of EAE. However, the underlying mechanisms and receptor location and subtype involved in cannabinoid-induced immunosuppression remains unclear. We demonstrate that amelioration of EAE by cannabinoids is associated with the suppression of antigen-induced T cell proliferation, pro-inflammatory Th1 (interferon gamma and tumour necrosis factor) cytokine production and inhibition of mononuclear cell infiltration of the central nervous system. Secondly, although leucocytes express CB 1 and notably CB 2 receptors, cannabinoid-mediated immunomodulation is largely, if not exclusively CB 1 receptor-dependent. Thirdly, using conditional CB 1 knockout mice, we demonstrate that the major pathway of immunosuppression is not due to CB 1 receptor stimulation on T cells but more likely the indirect stimulation of CB 1 in brain centres. Only high doses of cannabinoids that induced sedative physiological effects and these may not be clinically achievable or relevant. This suggests that cannabis use in humans is likely to exhibit no or marginal affects on relapsing disease in multiple sclerosis. OS2B-05 Fibrin/Mac-1 interactions induce microglia activation and regulate relapsing paralysis in central nervous system autoimmune disease In multiple sclerosis (MS), fibrin deposition temporally and spatially correlates with inflammatory demyelinating plaques. Although the presence of extravascular fibrin(ogen) at sites of inflammatory demyelination in MS has been documented by pathologists for decades, the molecular and cellular mechanisms of fibrin action in CNS pathogenesis have not been investigated. We show that fibrin signals through the Mac-1 (CD11b/ CD18) integrin receptor to activate microglia and regulate the progression and severity of inflammatory demyelination. Fibrinogen directly activates microglia resulting in dramatic cytoskeletal rearrangements and increased phagocytosis through the activation of Akt and Rho. Inhibition of fibrin/ Mac-1 binding with PI3K inhibitors (LY294002) or blocking antibodies inhibits fibrinogen-induced microglia activation and phagocytosis. Pharmacologic depletion of fibrin using the snake venom ancrod in remittingrelapsing Experimental Autoimmune Encephalomyelitis (EAE) results in reduced activation of CD11b-positive microglia cells and effectively reverses relapsing paralysis. Inhibition of fibrin/Mac-1 interactions in fibrin γ390-396A knock-in mice, as well as after vaccination or intranasal delivery of fibrin γ 377-395 peptide attenuates inflammatory demyelination. Our study provides a cellular and molecular definition of the role of fibrin in CNS pathogenesis and shows that fibrinogen-mediated microglia activation regulates the progression and severity of inflammatory demyelination. Since blocking fibrin/Mac-1 interactions affects only the inflammatory and not the coagulation properties of fibrin, targeting the γ 377-395 fibrin epitope could represent a potential therapeutic strategy for MS with potential applications for other neuroinflammatory diseases associated with blood-brain barrier disruption and microglia activation.Supported by NIH/NINDS R01 Grant NS052189 and NMSS RG 3782-A-2 (KA), and NMSS Postdoctoral Fellowship FG 1582-A-1 (RA). OS3A-01 The fourth nationwide survey of multiple sclerosis in Japan: MRI characteristics of Japanese multiple sclerosis Moreover, clinical characteristics were compared among OSMS with LESCL, OSMS with spinal cord lesion not extending over 3 vertebral segments (usual SCL), CMS with LESCL and CMS with usual SCL. Results: The frequency of patients who met the McDonald MRI criteria was significantly higher in CMS than in OSMS (45.5% vs. 8.2%, p < 0.0001), while the frequency of LESCL was significantly higher in OSMS than in CMS (41.2% vs. 16.1%, p < 0.0001). Regardless of whether CMS or OSMS, those with LESCL showed female preponderance, higher EDSS, higher frequency of paraplegia and higher CSF cell counts, compared with those having usual SCL. The EDSS of MS subgroups except OSMS with LESCL were significantly correlated with the disease duration. Regardless of whether LESCL or usual SCL, OSMS showed higher age at onset, lower frequency of secondary progression and higher frequency of negative Brain MRI, compared with CMS. Conclusions: The location of the lesions as well as the existence of LESCL is an important factor to decide a clinical feature of MS. OS3A-02 Clinical and laboratory features of neuromyelitis optica with oligoclonal IgG bands M. Nakamura a , I. Nakashima a , S. Sato b , K. Fujihara a , Y. Itoyama a a Departments of Neurology, Tohoku University School of Medicine, Sendai, Japan; b Department of Neurology, Kohnan Hospital, Sendai, JapanObjective: Oligoclonal IgG bands (OB) in the celebrospinal fluid (CSF) are an important laboratory finding for the diagnosis of multiple sclerosis (MS), and they are positive in more than 90% of MS patients in Western countries. In contrast, the frequency of OB has been reported to be much lower (about 10%) in neuromyelitis optica (NMO) even when sensitive isoelectric focusing is applied. The present study aimed to reveal the clinical and laboratory features of patients of NMO who were positive for OB.Method: We retrospectively reviewed the medical records of a total of 23 NMO patients (22 women and one man). We then analyzed the clinical data (age at onset, disease duration and Expanded Disability Status Scale (EDSS) score), CSF findings (cell count, protein concentration and IgG index) and immunological status (NMO-IgG and other autoantibodies, and co-existing autoimmune diseases) in the patients and compared the data of OB-positive patients with those of OB-negative patients. Both patients were also positive for NMO-IgG. Their common features were long disease durations and co-existing autoimmune diseases (myasthenia gravis and sicca syndrome). There was no difference in age at onset, EDSS score, CSF cell count and CSF protein concentration between the two groups. Conclusion: OB are mostly negative in NMO and helpful for distinguishing it from multiple sclerosis, but they can be positive by long-standing autoimmunity which may not be directly related to NMO. OS3A-03 Anti-aquaporin 4 antibody in Japanese opticospinal multiple sclerosis K. Tanaka a , T. Tani a , M. Tanaka b , T. Saida b , J. Idezuka c , K. Sakimura a , M. Nishizawa a a Brain Research Institute, Niigata University, Niigata, Japan; b Utano National Hospital, Kyoto, Japan; c Ojiya Sakura Hospital, Ojiya, Japan Neuromyelitica optica (NMO)-IgG, whose target molecule is probably an aquaporin 4 (AQP4), is reported to be a specific marker for NMO and will be a good tool to distinguish NMO from multiple sclerosis (MS). The differences between OSMS and NMO have long been under discussion. To clarify this argument, we established the immunofluorescence detection system of AQP4 antibody (AQP4-Ab) using AQP4-transfected HEK 293 cells. We examined this antibody in the sera of OSMS with long spinal cord lesions (LCL) with or without cerebral lesions, OSMS without LCL, common form of MS (CMS) and healthy controls. AQP4 was positive in 62% of OSMS with LCL but was negative in any other forms of MS. Among OSMS with LCL, the AQP4-positive group was all women with severe visual disturbance, however other features such as patients' mean age, severity of limb and truncal disabilities, existence of cerebral lesions were not different from AQP4-negative OSMS with LCL. Our results of AQP4-Ab in OSMS with LCL were similar in frequency of NMO-IgG found in NMO patients, indicated that both groups are immunopathologically common and could be the same. Japanese patients with MS who fulfilled Poser's criteria. All MRI scans were analyzed according to the McDonald/Barkhof's MRI criteria. We pathologically studied periventricular lesions in 3 opticospinal MS (OSMS) and 2 conventional MS (CMS) cases, as well as studying the features of LESCL. Results: The frequency of patients who met the McDonald/ Barkhof's MRI criteria and had periventricular ovoid lesions were significantly higher in CMS than OSMS patients (65.4% vs. 23.7%, p < 0.0001, 88.6% vs. 55.2%, p = 0.0026), while the frequency of LESCL and periventricular rim-like lesions were significantly higher in OSMS than CMS (55.3% vs. 25.0%, p = 0.0016, 62.1% vs. 31.4%, p = 0.0057). Pathologically, OSMS showed periventricular rim-like demyelinating lesions while CMS had demyelinating lesions extending to the deep white matter. LESCL distributed diffusely from the cervical to the thoracic cord, mainly involving the central portion of cord. Gadolinium-enhancing lesions were seen most frequently in the upper thoracic cord. Conclusion: The frequency of Asian MS patients fulfilling McDonald/Barkhof's MRI criteria is low while LESCL and periventricular rim-like lesions are frequently seen in OSMS patients. Introduction: The McDonald criteria of MS which is largely based on the features of Caucasian patients states the spinal lesion should not be longer than 2 vertebral length and refers to a paper describing such lesions were found in 2-3% of patients. In proposed criteria for neuromyelitis optica by Wingerchuk et al., long cord lesion extending over 3 vertebral segments (LCL) on spinal T2-MRI is the most important characteristic. We report here the frequent brain involvement in Japanese MS patients with LCL (LCL-MS). Methods: Among patients examined after 2000, we analyzed the clinical, MRI and laboratory features of 77 consecutive patients with LCL-MS. The average of disease onset age was 36 (2.5-74) years, disease duration 11.4 (1-37) years and the female to male ratio 8 to 1. Results: Site of clinical manifestation at the first attack was in spinal cord 54%, optic nerve 25%, both 6.8%, brainstem/cerebellum 15% and cerebrum 0%. In 98.6% cases, LCL was confirmed within 2 years after the onset. Cerebral symptoms, which were not present at the first attack, later developed in 39%: frequently symptoms were severe and the MRI lesion size was large. Brainstem lesions developed in 60%, 15% of whom required respirator assist. Average of recurrence number was 17.1 (in spinal cord 11.4, optic nerve 4.4, cerebrum 2.0, and brainstem/ cerebellum 1.4). Cerebrospinal fluid IgG oligoclonal bands were mostly negative. Conclusions: We propose LCL-MS as a variant of MS, at least in Japanese population, in which LCL occurs at an early stage and many later develop severe brain lesions. OS3B-01 Human myoblasts secrete MCP-1 and IL-6 and express ICAM-1 in response to IL-6/sIL-6R complex in vitro M. Marino a , F. Scuderi a , C. Provenzano a , Jürgen Scheller b , Stefan Rose-John b and Emanuela Bartoccioni a a Institute of General Pathology, Università Cattolica, Roma, Italy; b Department of Biochemistry, Christian-Albrechts-Universität zu Kiel, Kiel, GermanyMononuclear cell infiltration characterizes the inflammatory reaction in autoimmune polymyositis and rejection of transplanted myoblasts. Monocyte recruitment following neutrophil exudation is regulated by the IL-8/ MCP-1 switch driven by the action of the IL-6/soluble IL-6 receptor (IL-6/ sIL-6R) complex on endothelial/mesothelial cells. The IL-6R consists of a 130 kDa ubiquitously expressed transducing element, and a cytokinespecific 80 kDa receptor subunit; the latter is expressed only on target cells (hepatocytes and subsets of leukocytes) and is released as sIL-6R. The IL-6/ sIL-6R complex can interact with membrane-bound gp130 and activate virtually all cells in the body, through a mechanism called transsignaling. We have previously shown that human myoblasts are able to produce IL-6; the specific object of this study was to verify whether IL-6/sIL-6R complex could drive human myoblasts to substain monocyte recruitment. We used Hyper-IL-6 (a stable analogue of the IL-6/sIL-6R complex) to stimulate human myoblasts and analyzed the expression of MCP-1, IL-8, IL-6, membrane and soluble ICAM-1 and the monocyte chemotactic activity by RT-PCR, ELISA, FACS and chemotaxis assays.We found that human myoblasts do not produce significant levels of IL-8 in response to Hyper-IL-6, while they could use transsignaling to increase IL-6, MCP-1 and ICAM-1 production. Our findings show that muscle cells can use locally secreted IL-6 and transsignaling to start an autoamplificating autocrine IL-6 loop which then can target monocyte chemotaxis and leukocytes trafficking through the formation of an IL-6 and MCP-1 gradient and ICAM-1 modulation. OS3B-02 Expression of CCL19, CCL21/CCR7 chemokine system in muscles of polymyositis M. Tateyama, K. Fujihara, T. Misu, Y. Onodera and Y. Itoyama Department of Neurology, Tohoku University School of Medicine, Sendai, Japan Polymyositis is an autoimmune disorder in which autoaggressive CD8 + T cells are important in the pathogenesis. However, the mechanisms underlying sustained recruitment of these cells in the muscle tissue are still unknown. CCR7 and its ligands CCL19 and CCL21 are a chemokine system related to mononuclear cell migration and antigen presentation, and are suggested to play a key role in several autoimmune disorders. In immunohistochemistry, CCR7 was expressed mainly on mononuclear cells that infiltrated in the endomysium of polymyositis. 34.8 ± 9.4% of endomysial mononuclear cells expressed CCR7. By double immunostaining, about 60% of endomysial CD8 + T cells that infiltrated in endomysium and surrounded the nonnecrotic muscle fibers coexpressed CCR7. On the other hand, CCL19 was expressed mainly on muscle fibers in proximity to CCR7 + mononuclear cells, on the endothelium of the vessels and some mononuclear cells. CCL21 immunoreactivities were found on small numbers of mononuclear cells. In some cases, CCL21 immunoreactivities were also found on muscle fibers and the endothelium of vessels. In RT-PCR analysis, transcripts of CCR7 and CCL21 were detected in all the polymyositis muscles examined and that of CCL19 was detected in five out of seven polymyositis muscles. The CCL19,21/CCR7 chemokine system is expressed in inflamed muscles of polymyositis and may be involved in the pathomechanism of polymyositis.Magnetic resonance imaging (MRI) was investigated in 15 Japanese multiple sclerosis (MS) patients with NMO-IgG, a specific serum marker for neuromyelitis optica (NMO). The only patient without the longitudinally extensive spinal cord lesion, however, had extensive spinal cord atrophy indicating a history of an extensive cord lesion. Among the 7 patients with brain MRI lesions, 5 had only optic neuritis and transverse myelitis as clinical symptoms. The other 2 patients had no episode of optic neuritis but had histories of cognitive dysfunction, hemiparesis, or cerebellar ataxia. On brain MRI, diffuse white matter lesions, juxtacortical lesions, callosal lesions, periventricular lesions, and longitudinally extensive lesions along pyramidal tracts were commonly observed. Cerebellar hemisphere lesions were seen in one patient who developed cerebellar ataxia, and hypothalamic lesions were seen in another patient who showed hyperprolactinemia at the relapse of visual disturbance. Cavity formation was observed in none of the patients. Although some brain MRI lesions seen in the patients with NMO-IgG resembled those of the classic form of MS in shape, typical ovoid lesions with open-ring enhancement were not observed in any patient with NMO-IgG. Brain lesions were not rare in the patients with NMO-IgG, but the radiological features of the lesions were rather atypical for MS. OS3B-03 Molecular interactions between inflammation and β-amyloidassociated degeneration in sporadic Inclusion Body Myositis (sIBM) muscle Hallmarks of sIBM are inflammation and β-amyloid-associateddegeneration in the muscle, yet it is unknown whether these pathomechanisms trigger each other.RNA from sIBM-muscle (n = 12), Dermatomyositis (DM, n = 11), or non-myopathic controls (n = 12) was analyzed by real-time-PCR for expression of degeneration-associated-molecules amyloid-precursor-protein (APP), αB-crystallin, ubiquitin; inflammatory markers CXCL9, CCL3, CCL4, IFN-γ, IL-1β, IL-6, TNF-α, TGF-β, inducible-nitric-oxide (NO)synthase (iNOS). Magnetic-bead-purified human myotube-cultures were exposed to IFN-γ, TNF-α, IL-1β, or NO-donors, followed by analysis of cytokine-and chemokine-expression by real-time-PCR, immunohistochemistry and sandwich-ELISA. β-amyloid-accumulation was measured by FACS-analysis and immunohistochemistry with grayscale-densitometry.In sIBM-muscle compared to controls, mRNA for all chemokines (≤233-fold) and cytokines was significantly upregulated and half of the sIBM-samples displayed iNOS-expression. In DM, upregulated chemokine-mRNA (≤62-fold), TGF-β, and IL-6 remained significantly lower than in sIBM and only few patients expressed IFN-γ, IL-1β or iNOS. Degenerationassociated markers were significantly overexpressed in both disorders, yet only in sIBM, APP significantly correlated with mRNA-levels of chemokines, ubiquitin, IFN-γ, and cellular infiltration. By immunohistochemistry, chemokines and cytokines co-localized to β-amyloid-accumulation in sIBM-myofibers, whereas in DM, they were mainly present in connective tissue and capillaries. Inflammatory responses by myotubecultures were similar to the pattern observed in sIBM-muscle, including striking iNOS-overexpression after IL-1β + IFN-γ. Exposure to NO-donors or IL-1β + IFN-γ caused intracellular β-amyloid-accumulation as revealed by immunhistochemistry and FACS-analysis.In conclusion, the results suggest that a specific interrelationship between degenerative and inflammatory pathomechanisms in sIBM-muscle centers around NO-related cell-stress. The data further our understanding of sIBM-pathology and may help to better comprehend other β-amyloidassociated disorders. OS3B-04 Characterizing T cell receptors from human histological specimens S. Seitz ab , H. Wekerle a , R. Hohlfeld ab and K. Dornmair ab a Department Neuroimmunology, MPI for Neurobiology, Martinsried, Germany; b Institute for Clinical Neuroimmunology, LMU, Munich, GermanyWe describe a strategy to "revive" putatively pathogenic T cells from frozen specimens of human target organs. These T cells may then be used to investigate their yet unknown antigens. To distinguish autoaggressive from irrelevant T cells we focus on cytotoxic effector T cells that are in direct contact with their target cells and are clonally expanded as detected by immunohistochemistry and CDR3-spectratyping. Such cells were isolated by laser-microdissection from frozen biopsy specimens of polymyositis and inclusion body myositis lesions. We identified matching T cell receptor (TCR) αand β-pairs from several patients by a recently developed multiplex PCR protocol that allows concomitant amplification of both chains even from single T cells. From one patient we isolated 64 T cells which express an expanded Vβ1 chain. In 23 of these cells we identified matching α-chains, 20 of which were identical. We reconstructed this and several other paired TCR αand β-chains and expressed them functionally in T-hybridoma cells. Presently we are using these "revived" autoaggressive T cells to investigate their target antigens in vitro by cDNA expression and combinatorial synthetic peptide libraries. This protocol should be applicable not only to autoimmune diseases like multiple sclerosis, but also tumors and transplants. Background: To establish definite diagnoses of inflammatory myopathies including polymyositis (PM), dermatomyositis (DM) and inclusion body myositis (IBM), according to a recent criteria of the diagnoses, it is important to obtain muscle biopsy samples with inflammation. However, inflammatory change is often absent in the tissue obtained by conventional method of biopsy. Objective: To examine usefulness of magnetic resonance imaging (MRI) of skeletal muscles for determination of muscle biopsy sites to make diagnoses of inflammatory myopathies. Materials and methods: Method of MRI orientated muscle biopsy (MOMB) was established. Among 32 cases, excluding those already on steroids, which fulfilled criteria of Bohan and Peter for definite, probable or possible PM or DM and subjected to muscle MRI, 25 showed positive MRI change enabling us to apply MOMB, while 7 were negative on MRI. Muscle biopsy was performed in all these cases. For comparison, biopsies from 48 probable or definite cases of inflammatory myopathies, in which biopsy was performed at the belly of muscles before 2001. Results: By applying MOMB, number of samples with inflammatory change has increased significantly. On the other hand, in cases without significant change on MRI, chance to observe inflammation from MRI negative case was 29%. On the other hand, cases without significant change on muscle MRI emerged as a new issue. Method and patients: 30 CSF specimens from a group of healthy controls (HC) (5 samples) and MS patients (25 samples), classified as: Clinically Isolated Syndrome (7 samples), RRMS (7 samples), SPMS (5 samples) and PPMS (6 samples), were studied by means of the SELDI-tof. This technique allows the separation and identification by size and charge of a set of proteins in complex mixtures, as CSF.Results: The intensities of 13 potential differential mass-spectroscopy peaks associated to specific proteins or protein fragments are different between MS patients and healthy controls. The most significant differences were found between CIS and HC and PPMS and HC with 16 and 13 potential bio-markers, respectively. Despite of the reduced number of samples and therefore the limited statistical significance, it seems that two specific resonances assigned at 51,208 Da (p = 0.042) and 167,335 Da (p = 0.067) proteins could be considered as promising potential classifiers among the different MS clinical forms.Conclusion: The preliminary results obtained in this initial study seem to point out that SELDI-tof could be a potential useful technique to be applied in MS diagnosis by an adequate identification of specific proteins directly in CSF as discriminate bio-markers. More extensive studies using SELDI-tof are needed to verify the results obtained so far. OS4A-03 Bacterial peptidoglycan as a cofactor in multiple sclerosis inflammatory activity Peptidoglycan (PG) is a major bacterial cell wall compound with strong proinflammatory capacity. Membrane Toll-like receptor 2 is thought to be a signaling receptor for intact PG, while three types of innate receptors recognize small PG motifs intracellularly, namely Nod1/ CARD4, Nod2/CARD15 and NALP3/cryopyrin. Dysregulated function of these molecules is involved in several types of inflammatory disorders, including Crohn's disease and Blau syndrome. We previously proposed that PG is a factor codetermining inflammatory activity in multiple sclerosis (MS), and present three lines of evidence in support. First, in postmortem brain of MS patients, antigen presenting cells (APC) contain PG, presumably resulting from infiltrating cells which acquired PG fragments at the mucosa, and these APC are immunocompetent as evidenced by expression of costimulatory molecules and cytokines. Second, in two non-human primate experimental autoimmune encepha-lomyelitis models (marmoset and rhesus monkey), the burden of PG in the inflamed brain correlated with severity of pathology and APC subtype in the brain. Third, encephalitis could be precipitated in mice by adding PG as the sole microbial compound to incomplete Freund adjuvant plus encephalitogenic peptide. This collective immunopathological and functional evidence from rodent and non-human primate models, as well as from human MS, supports the potential role of PG as a cofactor in MS and warrants further elucidation of receptors and pathways involved. OS4A-04 Identification of novel biomarkers for multiple sclerosis based on the humoral immune response Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system in which both autoreactive T and B cells play a role. To fully explore the complex information present within the autoantibody repertoire of MS patients, we have applied a powerful procedure, called serological antigen selection (SAS), to identify antigens recognized by the humoral immune response in MS. The SAS procedure involves the display of a cDNA expression library from MS brain plaques on filamentous phage and subsequent selection on immunoglobulin G (IgG) present in serum or cerebrospinal fluid (CSF). To enrich for selection of cDNA products binding to IgGs specifically associated with MS, alternative selection strategies on MS patient material and normal samples were performed. Using SAS on pooled CSF (n = 10) from relapsing-remitting (RR) MS patients, we identified a panel of 8 different antigens, which after detailed serological analysis with 161 MS CSF and control CSF, showed exclusive or preferential reactivity to MS patient CSF. Affinity selections on a second pool of CSF from 10 other RR-MS patients resulted in the identification of 3 additional candidate antigens. In addition, selections on two pools of sera from 10 RR-MS patients each resulted in the identification of 20 cDNA clones. These clones are currently being further evaluated with regard to sequence identity and MS-specificity.In conclusion, our findings demonstrate that SAS is useful to identify novel candidate antigens in MS that can be used as diagnostic markers, and can be used to study the humoral immune response in MS. OS4A-05 IL-15 provided by myeloid cells and astrocytes in the human CNS contributes to the persistence of activated myelin specific CD8 T cells: Implications for multiple sclerosis CD8 T cells are prominent infiltrating cells in the central nervous system (CNS) during multiple sclerosis (MS). Highly activated memory CD8 T cells have been shown to clonally expand and persist in the CNS of MS patients for up to 5 years. Interleukin-15 (IL-15), a pro-inflammatory cytokine produced by macrophages and dendritic cells, is pivotal in the generation and maintenance of memory CD8 T cells. Higher amounts of IL-15 have been detected in MS patients' blood and cerebrospinal fluid. IL-15 bound to IL-15Ralpha on its own cell of origin, activates IL-15Rbeta/gamma bearing CD8 T cells. We sought to determine whether a local source of IL-15 can play a role in the acquisition of the memory phenotype by myelin specific CD8 T cells, and the persistence of such cells in the CNS of MS patients. Human monocytes/macrophages and astrocytes could provide the local source of IL-15/IL-15Ralpha since we observed that these cells express surface bound IL-15 up-regulated in response to pro-inflammatory cytokines. Myelin specific CD8 T cell lines cultured in the presence of IL-15 acquired a memory phenotype (CD45RO, CCR7-, CD62L-) that was not observed in the presence of IL-2. IL-15-cultured CD8 T cell lines contained high amounts of cytolytic enzymes, similar to activated NK cells. These results underline a novel potential role for IL-15 in the survival of memory myelin specific CD8 T cells in the CNS during inflammatory diseases such as MS. OS4B-01 CD11c on NK cells mirrors the disease activity of multiple sclerosis Toshimasa Aranami, Sachiko Miyake, Takashi Yamamura National Center of Neurology and Psychiatry, Kodaira, JapanMultiple sclerosis (MS) is an autoimmune disease, showing a great degree of variance in disease activity. We have recently demonstrated that NK cells biased for producing IL-5 (NK2 bias) are associated with remission state of MS. Here we report that upregulation of CD11c on NK cells would characterize a subgroup of MS in remission whose NK cells are lacking NK2 bias. When we compared this group (CD11c high ) with the other group of patients (CD11c low ) concerning NK cell expression of IL-5 and GATA-3, we found NK2 bias to be a selective character of CD11c low patients. In contrast, CD11c high group showed an obvious trend for a larger proportion of HLA-DR + cells within NK cells. Since NK cell stimulatory cytokine such as IL-15 was found to upregulate CD11c expression on NK cells in vitro, we postulate that inflammatory signals may play a role in inducing CD11c high NK cell phenotype. Although there was no clinical difference between CD11c high and CD11c low at blood sampling, CD11c high MS developed a clinical relapse significantly earlier than CD11c low during 120 day follow-up. These data point to an interpretation that CD11c high patients are at a higher risk for developing relapse, which is probably due to the loss of NK2 phenotype. Thus, CD11c on NK cells mirrors the disease activity of MS and may be used as a disease biomarker. Background: Plasmacytoid DCs (pDCs) represent a subpopulation of DC which exerts divergent functions in innate and adoptive immune response. It is thought that different DC-subtypes may be critically involved in the pathogenesis of multiple sclerosis (MS). Materials and methods: In our study we assessed by flow cytometry the phenotype of peripheral blood DCs from 35 clinically stable, untreated MS patients, 30 healthy controls and 9 patients with pneumonia used as nonspecific inflammatory conditions (NIC). For in vitro functional analyses DCs were isolated by magnetic sorting from leukapheresis material and cultured with trophic factors, unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides (CpG-ODN) or were used as regulators in co-culture system. Results: Ex vivo expression of CD86 and 4-1BBL was significantly lower on pDCs from MS patients than from controls and patients with NIC (22% vs. 47% vs. 41% and 12% vs. 35% vs. 32% respectively). In MS, pDCs stimulated with IL-3 and CD40L showed impaired maturation as demonstrated by significantly lower or delayed up-regulation of CD86, 4-1BBL, CD40 and CD83. Stimulation of pDCs by CpG-ODN resulted in a significantly lower IFNa secretion in MS than in controls. In MS but not in controls, pDCs failed to up-regulate proliferative responses and IFNg secretion of autologous PBMC in a co-culture system. Moreover depletion of pDCs in MS patients, but not in controls had no effect on generation of CD4 + Foxp3 + regulatory T cells. Conclusion: These findings suggest functional abnormalities of pDCs in MS patients, which might help to understand the mechanisms of immune dysregulation in this disease. In a search of reliable immunological parameters that can be used for assessment of disease activity, we examined paired blood and cerebrospinal fluid (CSF) samples obtained from 34 non-treated stable patients with relapsing-remitting multiple sclerosis (RRMS), who had been admitted for confirmation of diagnosis or to assess disease status. The samples were analyzed for humoral immunity as well as cellular immunity using flow cytometric examinations of CD4 and CD8 cells to determine their expression of surface antigens, including CD11a, CD25, CD26, CD29, CD45RA, CCR4, and CXCR3. Thereafter, the patients were followed for at least 1 year at the outpatient clinic, where they underwent neurological examinations as well as gadolinium-enhanced magnetic resonance imaging (MRI) scans, which were performed every 1 to 3 months until relapse became evident. Another group of 18 patients with noninflammatory neurological/psychiatric diseases served as controls. Of 27 RRMS patients who experienced a relapse within 1 year of the initial examinations, 7, 10, 6, and 4 showed relapse within 1, 3, 6, and 12 months, respectively. Those patients could be differentiated by the percentage of CD4 + CD25 + cells in the CSF. Patients with a greater than 3.6% ratio of CD4 + CD25 + cells in the CSF, which was 2 standard deviations above the mean of the controls, were more likely to relapse within 3 months. These findings suggest that enumeration of CD4 + CD25 + cells in the CSF can serve as a reliable measure of disease status in RRMS. However, the immunological relevance (regulatory or inflammatory) of these cells requires additional investigation. 0S4B-04 Altered naïve CD4 T-cell homeostasis in relapsing-remitting multiple sclerosis due to peripheral mechanisms Objectives: To investigate the involvement of peripheral mechanisms in homeostasis and to confirm our previous novel findings in RRMS. Methods: 19 untreated RRMS patients (mean age = 37.16) were recruited from the Montreal Neurological Hospital MS clinics. All patients had clinically definite MS based on McDonald criteria and were in clinical remission at the time blood was drawn. We recruited an independent set of 22 (mean age = 35) healthy controls, with no history of autoimmune disease. Lymphocytes were obtained using Ficoll-Paque™ and naïve CD4 T-cells were subsequently isolated by Macs® technology. Staining with PE-labeled anti-CD31 (indirect measure of homeostatic proliferation), PE-labeled anti-CD127 and PE-labeled anti-Bcl-2 (measures of homeostatic survival) and Facs™ analysis were performed in order to ascertain the percent expression of CD31, CD127 and Bcl-2 respectively. Serum IL-7 levels (measure of homeostatic survival) were determined by ELISA. DNA was extracted from the remaining isolated cells and signal joint T cell receptor excision circles (sjTRECsa surrogate marker of thymic production) were quantified using real-time PCR. Conclusions: Our observations confirm that there are indeed significant naïve CD4 T cell homeostatic abnormalities in RRMS and suggest that nonthymic peripheral mechanisms are involved. 0S4B-05 Identification of a pathogenic antibody response to native myelin oligodendrocyte glycoprotein in multiple sclerosis Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). Although the cause of MS is still uncertain, many findings point towards an ongoing autoimmune response to myelin antigens. We determined the serum antibody responses to native full-length MOG protein expressed on a human astroglioma cell line by a cell based assay. Antibodies, mostly IgG, were detected in serum of MS patients that specifically bound the extracelluar domain of MOG. These antibodies were directed against a conformational epitope that elicits a pathogenic antibody response in EAE. IgG but not IgM antibody titers to native MOG were significantly higher in the MS patient group compared to controls. One third of the MS patients, in particular primary progressive patients, had elevated anti-MOG antibody titers. These autoantibodies induced death of MOG-expressing target cells. Sera from MS patients with anti-MOG antibodies stained white matter myelin in rat brain and enhanced demyelination and axonal damage when transferred to EAE animals. 0S5A-01 Unexpected regulatory roles of TLR4 and TLR9 in an animal model for multiple sclerosis Innate immune mechanisms essential for priming encephalitogenic T cells in autoimmune neuroinflammation are poorly understood. To investigate the role of Toll-like receptors (TLR) and MyD88, the common TLR adaptor molecule, in an animal model of human multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) was induced by immunization with myelin oligodendrocyte glycoprotein (MOG) in complete Freund's adjuvant (CFA) in C57BL/6, TLR-or MyD88-deficient mice. MyD88 −/− mice were EAE resistant. However, we expected TLR4 −/− to have attenuated disease due to failure to signal the presence of TLR4binding pathogen associated molecular pattern (PAMPs) in mycobacteria, and similarly for TLR9 −/− mice due to failure to signal the presence of CpG DNA in mycobacteria. Instead, disease was paradoxically exacerbated in TLR4 −/− and TLR9 −/− mice. We also expected inhibited disease in TLR6 −/− mice due to failure to signal the presence of mycobacterial lipopeptides, but loss of this TLR did not affect the disease severity. MyD88 −/− mice failed to express IL-23 and IL-6, and serum and T cell IL-17 were absent. mDC IL-23 expression was higher and T cell and serum IL-17 levels were several-fold higher in TLR4 −/ − than in WT mice. In TLR9 −/− mice serum IL-17 levels were several-fold higher than in WT mice. Our data suggest that MyD88 mediates EAE via induction of mDC IL-23 and IL-6 that drives encephalitogenic T IL-17 cell activation. Data indicates a regulatory role for TLR4 and TLR9 in priming encephalitogenic T IL-17 cells. 0S5A-02 Redefining encephalitogenicity: Identification of a novel pathogenic cytokine induced by IL-23 K. Kreymborg a , R. Etzensperger b , S. Haak a and B. Becher a a Department of Neurology, Universitatsspital/University of Zurich, Frauenklinikstrasse 10, CH-8091 Zurich, Switzerland; b Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, UK Tissue-directed autoimmune diseases were widely held to be mediated by the conduct of self-reactive Th1 cells. However, we have previously shown that neither Th1 nor Th2 gene-expression serves as a reliable marker for pathogenicity. Furthermore, the master-cytokine Interleukin (IL)-23 was shown to be absolutely critical for the induction of autoimmune disease as demonstrated in collagen-induced arthritis (CIA) as well as experimental autoimmune encephalomyelitis (EAE). It becomes increasingly evident that IL-23 supports the expansion of a distinct lineage of CD4 + cells that produce IL-17 (Th IL-17 cells) and play a major role in autoimmune inflammation. We discovered an IL-10 related cytokine whose expression profile is identical with that of the disease associated IL-17 but hasin contrast to IL-17so far never been described in the context of EAE. Our findings point towards a major role of this illdescribed cytokine. Taken together, this molecule will help to define the encephalopathogenic phenotype of autoaggressive effector cells and permits the precise characterization of this novel population of Th IL-17 cells. 0S5A-03 Over-expression of CD24 in the central nervous system leads to enhanced disease progression in experimental autoimmune encephalomyelitis Jin-Qing Liu, Pramod S. Joshi, Joe Carl, Xue-Feng Bai Department of Pathology, The Ohio State University Medical Center, Columbus, OH 43210, USA Experimental autoimmune encephalomyelitis (EAE) is an experimental model of human disease multiple sclerosis (MS). The development of EAE requires CD24 expression on both T cells and local cells of the CNS. In order to understand the role of CD24 expressed on local CNS cells during EAE development, we generated CD24 transgenic mice in which CD24 was expressed in the CNS under the control of the GFAP promotor (Astro CD24TG mice). Upon immunization, Astro CD24TG mice developed a more progressive disease compared with their non-transgenic littermates (NTG), which was reflected by persistent EAE signs and large areas of demyelination and axonal damage in the spinal cords at later stages of EAE in Astro CD24TG mice. The enhanced EAE progression was associated with higher expression of cytokine genes including IFNγ, IL-17, TNFα and IL-10 but not IL-4 in the spinal cords of Astro CD24TG mice than that of NTG mice. Moreover, expression of a demyelination associated marker P8 was also increased in the spinal cords of Astro CD24TG mice compared with NTG mice. Finally, adoptively transferred 2D2 T cells, which are specific to MOG p35-55, expressed a more mature effector phenotype in the CNS of Astro CD24TG mice than in NTG mice. Thus, over-expression of CD24 in the CNS enhances EAE development via enhanced co-stimulation of encephalitogenic T cells. Since CD24 is drastically increased both on local cells and inflammatory cells in the CNS during EAE, our data have important implications for CD24 targeted therapy of MS. EAE is an animal model of multiple sclerosis and regulatory T cells play an important role in modulating EAE and in recovery. We have been studying TGF-β dependent regulatory T cells that are associated with recovery from EAE and that can be induced following oral administration of antigens and more recently oral anti-CD3 monoclonal antibody (Nature Medicine, in press). We have found that the surface expression of latency associated peptide of TGF-β (LAP) marks a T cell with regulatory properties and that CD4 + LAP + T cells increase in the spleen and peripheral blood of animals during the EAE recovery phase. We have now initiated studies of CD8 + LAP + T cells. CD8 + CD25 − LAP + and CD8 + CD25 + LAP + cells from spleens and lymph nodes of SJL mice isolated by high speed cell sorting were anergic when they were stimulated with anti-CD3 in the presence of APC in vitro. Furthermore, these two populations suppress the proliferation of responder T (CD4 + CD25 − LAP − or CD8 + CD25 − LAP − ) cells in vitro. We also found that CD8 + LAP + T cells produce more TGF-β and significant higher amount of IFNγ compared with CD8 + LAP − T cells. Thus, in addition to CD4 + LAP + T cells, CD8 + LAP + T cells may play an important role in the regulation of EAE. The secretion of IFNγ may also suggest a role for CD8 + LAP + T cells as a regulatory T cell in allergy. 0S5A-05 PSGL-1 and Fucosyltransferase-VII are involved in the suppressor activity mediated by CD4 + CD25 + regulatory T cells The mechanisms involved in the regulation of experimental autoimmune encephalomyelitis (EAE) by CD4 + CD25 + regulatory T cells (Tregs) are not well understood. The goal of this study was to determine the role of Alpha (1,3) fucosyltransferases (FucT) and P-selectin glycoprotein ligand-1 (PSGL-1) in the suppression of EAE by Tregs. Methods: Active and transfer EAE models using MOG 35-55 peptide were performed in WT/ C57Bl/6, FucT-VII −/− , FucT-IV −/− and PSGL-1 −/− mice. Isolation of CD4 + CD25 + Tregs, flow cytometry, adhesion and proliferation assays were also performed.Results: FucT-VII −/− and PSGL-1 −/− mice developed a more severe active EAE when compared to wt animals. Moreover, encephalitogenic T cells produced from FucT-VII −/− and PSGL-1 −/− mice transferred a significantly more severe disease that wt T cells. We observed no significant differences between the expression of adhesion molecules and IL-4 and IFNgamma production of autoreactive T cells from PSGL-1 and FucT-VII deficient mice. However, co-cultures with CD4 + CD25 + Tregs and effector cells showed that deficiency of PSGL-1 and FucT-VII leads to a marked decrease of suppression capacity of Tregs. Moreover stimulation of Tregs with anti-CD3 and IL-2 induces an increase of adhesion capacity with an increase expression of PSGL-1 and an increase binding of P-and E-selectin chimeras. Importantly, stimulated CD4 + CD25 + Tregs display increased expression of functional PSGL-1 and a significantly higher suppressor activity.Conclusion: Our data show that PSGL-1 and FucT-VII are involved in the immune suppression mediated by CD4 + CD25 + Tregs in MOG-induced EAE, and suggest that in vitro manipulation of Tregs may lead to increased suppressor activity in inflammatory diseases.The immunological synapse forms in the contact zone between two immune system cells and may optimise activation by congregating signalling molecules in specialised regions of the plasma membranes; however, its precise functions remain a matter of debate. It has become clear that L-glutamate, the major excitatory neurotransmitter plays a role in peripheral tissues, including the immune system. Here we show that Lglutamate, a major excitatory amino acid in synaptic transmission in the nervous system, acts as an immunotransmitter at the immunological synapse. Dendritic cells (DCs)the most potent antigen-presenting cells contain a vesicular compartment competent for regulated glutamate exocytosis. They also express glutamate-specific vesicular VGLUT transporters conferring a glutamatergic phenotype on neurons and release glutamate through Ca 2+ -dependent exocytosis. Remarkably, thymocytes express a large repertoire of glutamate receptors (GluRs), mirroring that of neural cells. In particular, they express functional NMDA receptors (NMDARs), which contribute to the Ca 2+ signal leading to antigendependent apoptosis in the context of T-DC synapses, mimicking thymic negative selection. We have identified the prototypical post-synaptic PDZ domain protein PSD-95 as a new marker of the immunological synapse. PSD-95 is recruited in the T-DC contact zones where it colocalizes with NMDARs. T cell stimulation, even in the absence of glutamate, also results in the co-localisation of PSD-95 with the NMDAR in the contact zone, suggesting that T-cell receptor triggering is involved in the recruitment of functional NMDARs at the synapse. This work reveals a new aspect of immunological synapse functioning, in which, similar to central nervous synapses, DC-released glutamate elicits focal responses in T cells bearing GluRs. Glutamate should therefore be considered a synaptic transmitter, acting through fine-tuning of Ca 2+ mobilization, in both the nervous and immune systems. AM is a novel peptide highly expressed by cerebral endothelial cells. In vitro studies show the peptide to regulate key BBB features including transendothelial resistance and permeability. Furthermore, we have shown that AM is elevated during EAE when the BBB is dysfunctional and inflammatory lesions apparent. AM acts primarily through the calcitonin receptor-like receptor (CRLR) in combination with either receptor-activity-modifying-protein (RAMP) 2 or 3. The main cellular target for AM during neuroinflammation is uncertain. We have therefore assessed receptor expression on T-cells plus component cells of the BBB.RAMP2 and 3 and CRLR expression was examined using PCR and FACS techniques. We found activated T-cells predominantly expressed RAMP3, although some RAMP2 was also present, with a significant proportion sited on the membrane. Conversely, for both ECV304 human endothelial and C6 rat astroglioma cells, the majority of the RAMP2 and 3 was located in the cytoplasm, with predominantly RAMP2 expressed under physiological conditions. CRLR was present in all cell types examined. Interestingly, the response of RAMPs to glucocorticoid exposure (10 − 6 -10 − 8 M) differed dependent on cell type, indicating that AM could have an important role in management of neuroinflammation. The peptide could therefore be involved in the neuroinflammatory processes evident in human conditions such as multiple sclerosis. 0S5B-03 Role of capsaicin-sensitive sensory nerves and the TRPV1 capsaicin receptor in endotoxin-induced airway inflammation and hyperreactivity in mice Capsaicin-sensitive, transient receptor potential vanilloid 1 (TRPV1) receptor-expressing, primary sensory nerves play important regulatory role in the pathomechanism of several inflammatory processes. The aim of the present study was to examine the role of these afferents and TRPV1 receptors in endotoxin-induced airway inflammation and hyperreactivity in mice. Pneumonitis was evoked by intranasal E. coli lipopolysaccharide (LPS) and enhanced pause (Penh), a calculated parameter correlating to airway resistance, was measured by whole body plethysmography. Bronchoconstriction was induced by inhalation of the muscarinic receptor agonist carbachol 24 h after LPS. Histological scoring, measurement of myeloperoxidase activity referring to granulocyte accumulation and determination of the interleukin-1β were performed from the lung. For the inactivation of capsaicin-sensitive afferents resiniferatoxin (RTX) pretreatment was performed, the function of the TRPV1 receptor was examined using gene-deleted (TRPV1 −/− ) mice.Destroying capsaicin-sensitive afferents by RTX-desensitization markedly enhanced all the inflammatory parameters, but inhibited hyperreactivity supporting the concept that tachykinins directly contribute to bronchoconstriction. Meanwhile, the lack of the TRPV1 receptor increased both inflammation and airway hyperresponsiveness. Specific radioimmunoassay was used to identify the inhibitory mediator released from sensory fibres via TRPV1 activation and it was proved to be somatostatin. LPS increased somatostatin in the lung and plasma in TRPV1 +/+ , but not in TRPV1 −/− mice. The somatostatin receptor antagonist cyclo-somatostatin aggravared inflammatory parameters and hyperresponsiveness pointing out its functional significance.These results provide the first evidence for a novel inhibitory mechanism mediated by somatostatin derived from sensory afferents via TRPV1 receptor activation in the airways.Grants: OTKA F-046635; ETT03543/2003; RET-008/2005. 0S5B-04 Compartmentalized immune response in the barrel field of the mouse primary somatosensory cortex The brain is constituted by modular units that group nervous cells and connections that process a similar type of neural information. On the other hand, previous observations in patients with neurocysticercosis showed that the immune response differs among brain regions, thus suggesting the existence of immunological compartments in the nervous system. Whether immune compartments and neural modules share their location and function has not been addressed. To explore this possibility we studied the immunological response to lesions carried out within and outside neural modules located in the primary somatosensory cortex of adult mice. These modules are called barrels and they represent collections of mechanosensory receptors. We found that the neural tissue adjacent to lesions placed outside the barrel field showed an intense astrocytic response and MHCII, INFγ, TNFα immunoreactivity in macrophages and activated microglia. These cells also expressed IL4 and IL10. In contrast, lesions placed inside the barrel field displayed MHCII, IFNγ and TNFα immunoreactive microglia and macrophages predominantly in the border of barrels right next to the lesion. There was an extremely reduced response in the barrel center, as it was in barrels close and far from the lesion site. Our observations open a new perspective for the study and understanding of the physiology and the pathology of the CNS. Systemic inflammation, impacts on the brain and gives rise to behavioural changes, often referred to as 'sickness behaviour'. These symptoms are thought to be mainly mediated by pro-inflammatory cytokines. Here we study communication pathways between the immune system and higher brain function following sub-pyrogenic inflammation.Systemic inflammation was induced in mice using models of bacterial (Lipopolysaccharide (LPS); 1-100 μg/kg) or viral infection (poly I:C; 12 mg/kg), and changes in open-field activity, burrowing behaviour and consumption of glucose solution were assessed. Immune activation was studied in the periphery and brain by measuring cytokine production, and immunohistochemistry.Sub-pyrogenic inflammation resulted in changes in species-typical, untrained behaviours that depend on the integrity of the hippocampus. Increased expression of cytokines was observed in the periphery and selected regions of the brain which coincided with behaviour changes. However, after neutralization of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-a), no significant differences were found when compared to LPSplus-vehicle treated mice. In contrast, pre-treatment of mice with indomethacin completely prevented LPS induced behaviour changes, without affecting cytokine levels.Our work, studying the effect of low grade inflammation on affective behaviour in mice, suggests a key role for prostaglandins, rather than cytokines, in communicating changes in immune status to the brain. This study was sponsored by BBSRC and UCB Celltech. Neurogenesis is known to continuously take place in certain 'neurogenic' areas of the adult central nervous system (CNS) and can be induced in 'non-neurogenic' areas under traumatic or degenerative conditions. Here we introduce T cells and CNS-resident microglia as important players in the regulation of adult neurogenesis. Under normal conditions, immune deficient mice (SCID and nude) and transgenic mice that most of their T-cell pool is specific for an irrelevant antigen (ovalbumin) exhibited impaired hippocampal neurogenesis. In contrast, mice in which the majority of T cells specifically recognize the CNSabundant antigen myelin basic protein showed normal neurogenesis. CNSspecific T cells were also found to be important for spatial learning abilities and for brain-derived neurotrophic factor expression in the dentate gyrus. Environmental enrichment did not evoke enhanced neurogenesis in immune-deficient animals, whereas in wild-type animals it led to enhanced hippocampal neurogenesis coupled with recruitment of T cells and activation of microglia. The clinical implications of these findings were tested using a rat model of cerebral ischemic insult. We demonstrate that Tcell based immune activation following stroke induces a robust elevation of neurogenesis in the hippocampus as well as in the cerebral cortex. Our results suggest that T cells, acting via resident antigen presenting cells, are important regulators of adult neurogenesis under both physiological and pathological conditions. 0S6A-02 Immune cells contribution to hippocampal plasticity is age-dependent, and correlates with local modulation of gene expression Hippocampal neurogenesis is known to take place throughout adult life and is considered necessary for certain types of cognitive abilities. We have lately demonstrated that peripheral immunity contributes to adult hippocampal neurogenesis and spatial learning/memory, and that this is mediated in part by T lymphocytes recognizing brain antigens such as myelin proteins. Here we investigated whether CNS specific T cells contribute to the maintenance of brain plasticity only at adulthood, or whether they are required at earlier stages as well. We examined hippocampal neurogenesis in wild-type mice and in transgenic mice that express a T-cell receptor that recognizes ovalbumin ('T OVA -transgenic' mice), and thus bear mainly T OVA cells and no autoimmune cells, at different ages. Interestingly, the difference between wild-type and T OVA -transgenic mice in terms of the number of newly formed neurons generated in the hippocampus was relatively small at infancy, increased at adulthood and decreased for older age. We searched for hippocampal expressed genes, which were possibly controlled by immune cells. Although 'T OVA -transgenic' mice differ from their controls only in their peripheral T cell repertoire, significant differences in expression of several hippocampal genes were found. Among the differentially expressed genes we found some that were known to be involved in neurogenesis and axonal guidance. Further analysis of such genes will contribute to understanding how adult neurogenesis is controlled. 0S6A-03 Role of T cells in toluene-induced memory-related gene expressions in mouse hippocampus Recently, it has been demonstrated that lack of mature T cells in mice manifested cognitive deficits and which could be restored by passive T cells transfer. Toluene mainly affects the central nervous system causing memory loss. Toluene may produce some of its effects by directly inhibiting Nmethyl-D-aspartate (NMDA) receptor function in a subunit-selective manner. To investigate the possible involvement of T cells in toluene-induced expression of memory-related gene in mouse hippocampus, we exposed BALB/c and nude mice to toluene at a low dose (9 ppm), and to air control (0 ppm toluene) as well in nose-only exposure chamber for 30 min in 3 consecutive days followed by once a week for 4 weeks. Four hours after the last challenge, we collected the hippocampus and examined the mRNA expression of NMDA receptor, protein kinase and transcriptional factors, which are potentially involved in memory functions, and then chemokines by a quantitative real-time PCR method. Our data clearly demonstrates that NMDA receptor NR2A, Ca 2+ /calmodulin-dependent kinase, CaMKIV, and transcriptional factor cAMP responsive element-binding protein, CREB-1, CCL2 and CCL3 mRNA expressions increased in the hippocampus of BALB/c mice exposed to 9 ppm toluene compared to that of air control. After immunization with ovalbumin, BALB/c mice showed no difference of NR2A and CaMKIV mRNA expression between air control and toluene (9 ppm). There were no alterations observed in nude mice with or without immunization. These findings indicate that T cells may play a role in memory-related gene expression induced by toluene exposure in BALB/c mice. 0S6A-04 Non-MHC genes regulate locomotor behavioral outcome following rat spinal cord contusion injury Secondary degeneration resulting from spinal cord injury is characterized by inflammation and progressive loss of nervous tissue. In order to dissect this process genetically and to examine potential influence by the MHC locus, known to be of importance for regulation of autoimmune processes, a well-characterized weight-drop contusion model was performed in a panel of inbred strains with different susceptibility to neuroinflammation. Included strains were: ACI, DA, LEW and PVG, as well as the MHC congenic strains LEW.1AV1, LEW.1N, LEW.1W and PVG.1AV1. Striking differences in motor behavioral outcome across the strains were observed. Interestingly, the best outcome was demonstrated for the inflammation prone DA rat, whereas the PVG strain, resistant in most inflammatory models, displayed an intermediate response. The different LEW strains, the wild type and three strains congenic for different MHC haplotypes, displayed the same worst outcome. No MHC haplotypedependent effects were observed in a F2 intercross experiment (PVG × PVG.1AV1), providing additional evidence for a dominant influence of non-MHC genes. We conclude that outcome following spinal cord injury is subject to considerable genetic heterogeneity. However, this genetic regulation does not seem to depend on the degree of susceptibility to autoimmune neuroinflammation or, in particular, the MHC complex. 0S6A-05 The protective role of autoimmune reaction in the neurodegenerative process caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropiridine (MPTP) in mice The response of the immune system during injury of the central nervous system has possible protective role. Here we present the influence of the autoimmune reaction on dopaminergic neurons damage induced by MPTP. C57Bl male mice, 2 (young) and 10 (aged) months old were injected with MPTP (40 mg/kg). One group of each age, 6 days before the injection, had induced autoimmune encephalomyelitis (EAE) by myelin oligodendrocyte glycoprotein (MOG) 35-55 in CFA administration. In these mice, MPTP caused less dopaminergic system damage than in MPTP only injected mice. The dopamine level was higher by 10% and 30% and the number of preserved neurons by 16% and 21% in young and aged animals respectively, on the 7th day after MPTP administration. The microglial reaction was diminished and lymphocytic CD8 + infiltration significantly reduced. TGF and GDNF mRNA expressions were significantly higher in MOG receiving mice when compared to MPTP group. We observed also shift in T cells populations, from the preponderance of CD8 + in MPTP group to CD4 + in MOG group. We injected mice with CD4 + anti-MOG T cells a day after MPTP administration and we observed also less damage of the dopaminergic cells when compared to MPTP only injected mice. We showed that immune enhancement is protective in toxic damage of the central nervous system. CD4 + cells are possibly involved in the protective mechanism, but the induction of trophic factors by other factors is also the option. An association between the autoimmune disease multiple sclerosis and HHV-6A has been suggested. In vivo, HHV-6A has been detected in myelin producing cells, i.e. the main cells that are affected in MS. HHV-6A might incorporate host cell proteins into the viral particles during replication. When the virus is detected by the immune system, the incorporated proteins will be presented for the immune system. This could be one mechanism for induction of autoimmunity.Incorporation of host cell proteins have been shown for other viruses, for example the complement proteins CD55 and CD59 that are incorporated into HCMV, HTLV-1 and HIV-1. However, the purity of the virus preparations has been debated since cellular vesicles might contaminate the viral preparations during purification in sucrose gradients. In this study, we used iso-osmotic iodixanol gradients to purify T-cell cultured HHV-6A. In our gradient peak fraction we detect high levels of viral DNA by real-time PCR, expression of gp60/110 by Western blot and intact viral particles by electron microscopy. The purified virus particles were also able to re-infect T cells with good efficiency. We have low cellular contamination in the viral particle containing fractions as seen by analyzing viral and mock preparations by SDS-PAGE. Immunoblotting with anti-CD46 and other anti-host cell protein antibodies gave clear bands in the viral containing fractions compared to the mock containing fraction.In conclusion, this method may be used to study the protein content of viral particles and that may give important clues for how autoimmunity is induced. Peripheral neuropathy is the most common neurological complication associated with HIV-1 infection, affecting over one-third of individuals with AIDS. Studies also have found a high prevalence of HIV sensory neuropathy in HIV-infected patients despite administration of highly active antiretroviral therapy. To study HIV-induced PNS disease, we established an SIV/macaque model in which over 90% of animals developed PNS changes closely resembling those seen in HIV-infected individuals, including inflammation of the dorsal root ganglia with abundant replication of SIV in macrophages and DRG neuronal loss, sural nerve inflammation, and reduction in epidermal nerve fiber density in distal extremities. This constitutes the first primate model of HIVinduced peripheral neuropathy. In this SIV model we examined the relationship between dorsal root ganglia (DRG) changes and sensory nerve function by measuring C-fiber conduction velocities in the sural nerves and epidermal nerve fiber density in the skin of SIV-infected macaques and uninfected control macaques. SIV-infected macaques had significantly lower sensory C-fiber conduction velocity (CV) in sural nerves than uninfected animals (p = 0.01). The extent of conduction velocity decline correlated strongly with extent of macrophage infiltration in the DRG (p = 0.006). Significant declines in density of epidermal nerve fibers also developed in SIV-infected macaques (p = 0.01) but were not strongly correlated with changes in the CV. These findings suggest that primary injury to the neuronal cell body in the DRG mediated by activated macrophages alters functional properties of sensory nerves in HIV-infected individuals. 0S6B-03 Efficacy of intracerebral vaccination against pseudorabies virus in mice Jae-Ho Shin 1,2 , Yoshihiro Sakoda 3 , Jae Hoon Kim 1, 4 To evaluate the efficacy of intracerebral (IC) vaccination, mice were immunized with formalin-inactivated pseudorabies virus (PRV) by either subcutaneous (SC) or IC injection, and then 10 6 plaque-forming units of PRV were introduced into the hindleg of the immunized or non-immunized mice by intramuscular injection. The antibody titer in serum was elevated and boosted by additional vaccination via both the SC and IC route, but was higher after IC vaccination. Intracerebrally immunized mice were completely protected from mortality and neurological signs, whereas all the non-vaccinated and 80% of the subcutaneously vaccinated mice died after developing neurological signs. IC vaccination is more effective at inducing a protective immune response against the transneural spread of virus than a SC vaccination.Keywords: Intracerebral Vaccination; Transneural Spread; Pseudorabies Mouse. 0S6B-04 Role of dendritic cells in the transmission and induction of neuroinvasion of scrapie following intravenous infection Claudine R Raymond, Moira E Bruce and Neil A Mabbott Institute for Animal Health, Neuropathogenesis Unit, Ogston Building, West Mains Road, Edinburgh, UK, EH9 3JF. ac.uk Following peripheral scrapie inoculation, the agent accumulates on follicular dendritic cells (FDCs) in lymphoid tissues before spreading to the brain. How TSE agents reach lymphoid tissues and the nervous system following exposure is not known. Recent evidence suggests possible roles for dendritic cells (DCs) and macrophages, as disease-specific prion protein (PrP Sc ) has been found associated with each of these cell types following exposure to scrapie. The current study utilized two intravenous infection models of scrapie (ME7 and 139A agent strains) to determine if scrapie-infected mononuclear cells (MNCs), especially DCs and B cells, could deliver infection directly to the nervous system in the absence of FDCs and other lymphoid elements. Scrapie-infected MNCs, B cells and DCs efficiently transferred infection when transfused into wild-type recipients. Indeed, the disease incubation period following transfusion of scrapie-infected DCs was similar to that of the MNCs and B cells even though 10-20× fewer cells were used. However, in the absence of FDCs in TNF-receptor-deficient mice, the ability of the scrapie-infected cells to transmit infection was greatly reduced. These results demonstrate that for both the ME7 and 139A strains of scrapie, efficient neuroinvasion from the periphery requires prior agent replication/ accumulation on FDCs. In the absence of this accumulation, DCs and other mononuclear cells capable of delivering the scrapie agent directly to the nervous system but via a substantially less efficient mechanism.Funded by EU grant no. 0S6B-05 Regulation of the inflammatory response to Staphylococcus aureus-induced brain abscess by interleukin-10 A characteristic of brain abscess is a localized suppurative infection leading to substantial damage of the adjacent CNS tissue. The orchestrated interplay of pro-and anti-inflammatory cytokines released by leukocytes as well as resident cells of the central nervous system is crucial for both an effective host defense, and for limiting tissue damage in brain abscess. To study the regulatory role of interleukin (IL)-10 in brain abscess in vivo, IL-10 deficient (IL-10 0/0 ) mice were stereotaxically infected with Staphylococcus (S.) aureus-laden agarose beads. Increased numbers of intracerebral (i.c.) granulocytes, macrophages, CD4 + and CD8 + T cells and higher levels of TNF, IL-1β, and iNOS were observed in IL-10 0/0 mice than in wild type mice, while chemokines were induced earlier and more pronounced in WT mice. Together with prominent microvascular haemorrhage, necrotic vasculitis, severe brain edema, and markedly increased abscess size, these alterations led to an increased morbidity of IL-10 0/0 mice. Nevertheless, the hyperinflammatory response of IL-10 0/0 mice did not improve bacterial elimination. host immune response in experimental S. aureus-induced brain abscess.Oral Session 7A: Immunotherapy (1) 0S7A-02Glatiramer acetate mediated immune modulation is driven by antigen presenting cells and is not T cell antigen specific Background: GA exerts therapeutic benefit in multiple sclerosis presumably by induction of GA-specific Th2-cells. Recent reports revealed that GAtreatment alters APC function. We investigated (1) whether these APC effects mediate T-cell immunomodulation, (2) whether GA-treated monocytes can adoptively transfer protection from experimental autoimmune encephalomyelitis (EAE) and (3) whether T-cell cross-reactivity between GA and myelin antigen is required for its therapeutic effect. Methods: (PL/JxSJL/J)F 1 mice were injected subcutaneously with 150 μg GA/d in PBS prior to or after EAE onset. T-cells were cultured with GA, intact MBP, MOGp35-55 or PLP139-151. Monocytes from GA-treated wild-type or RAG-1-deficient mice were co-cultured with naive T-cells from MBP Ac1-11 TCR transgenic (Tg), MOG35-55 TCR or OVA323-339 TCR Tg mice. GA-treated monocytes were injected into immunized C57BL/6 mice. Results: Daily GA administration without adjuvant prevented or reversed EAE. We did not observe T-cell cross-reactivity between GA and myelin antigens. GA treatment of wild-type and RAG-1-deficient mice, which lack mature T-cells, induced type-II APC, secreting less TNF-a, IL-12 and more IL-10. These APC directed Th2-differentiation of naive myelin-and OVAspecific T cells, induction of regulatory T cells (Treg) and protected recipient mice from EAE. Conclusions: Therapeutic benefit of GA in EAE does not require T-cell crossreactivity between GA and myelin antigen. GA-treatment exerts a direct effect on APC without the requirement of T-cells. GA-mediated APC immunomodulation drives Th2-polarization and Treg-induction independent of T-cell specificity and is sufficient to adoptively transfer EAE protection. 0S7A-03 Suppression of ongoing disease in a non-human primate model of multiple sclerosis by a human-anti-human IL12p40 monoclonal antibody IL-12p40 is a shared subunit of two cytokines with overlapping activities in the induction of autoreactive Th1-cells and therefore a potential target of therapy in Th1-mediated diseases. We have examined whether ongoing disease in a non-human primate model of multiple sclerosis can be suppressed with a new human IgG1κ mAb against human IL-12p40. Lesions developing in the brain white matter were visualized and characterized with standard magnetic resonance imaging (MRI) techniques. To reflect the treatment of MS patients, treatment with the mAb was initiated after active brain white matter lesions were detected in T 2 -weighted images. In placebo-treated control monkeys we observed the expected progressive increase of the total T 2 lesion volume and markedly increased T 2 relaxation times, an MRI marker of inflammation. In contrast, in monkeys treated with anti-IL-12p40 mAb changes of the total T 2 lesion volume and T 2 relaxation times were significantly suppressed. Moreover, the time interval to serious neurological deficit was delayed from 31 ± 10 days to 64 ± 20 days (odds ratio 0.312). These results in a disease model with high similarity to MS are important for ongoing and planned trials of therapies that target IL-12 and/or IL-23. Background: Interferon beta (IFNβ) is the most commonly used immunomodulatory treatment for MS and is administered to patients as an exogenous recombinant protein. Microbial components can induce the production of endogenous IFNβ by antigen-presenting cells (APC) through stimulation of TLRs. Poly I:C is a mimic of double-strand RNA that stimulates APC through TLR3. Methods: Female SJL mice were immunized with proteolipid protein (PLP) peptide (PLP) 139-151 and treated with poly I:C or PBS injected intraperitoneally during the induction or the relapsing phase of disease. Clinical, pathological, and immunological parameters were assessed. Poly IC induced significantly increased production of the CC-chemokine CCL2 (MCP-1) by spleen cells cultured ex vivo in the presence of PLP 139-151 . Neutralization of either IFNβ or CCL2 in vivo by injection of neutralizing antibodies reversed disease suppression by poly I:C. In vitro neutralization studies in bone marrow-derived dendritic cells showed that the production of CCL2 was IFNβ-dependent. The induction of endogenous IFNβ will be further assessed as a strategy for the treatment of MS. 0S7A-05 Selective COX-2 inhibitor celecoxib prevents experimental autoimmune encephalomyeritis through COX-2 independent pathway Katsuichi Miyamoto a , Sachiko Miyake b , Susumu Kusunoki a , Takashi Yamamura b a Department of Neurology, Kinki University, Osaka, Japan; b Department of Immunology, National Institute of Neuroscience, NCNP, Tokyo, Japan Cyclooxygenase (COX) is a key enzyme of arachinoic acid metabolism and exist as two distinct isoforms. COX-1 is constitutively expressed in most tissues, whereas COX-2 is inducibly expressed at site of inflammation. Selective inhibitors of COX-2 have been developed and have been used as anti-inflammatory agents. Here we show that a new generation COX-2 inhibitor, celecoxib inhibited experimental autoimmune encephalomyelitis (EAE). Celecoxib but not other COX-2 inhibitors such as nimelsulid prevented myelin oligodendrocyte glycoprotein (MOG)-induced EAE when administrated orally on the day of disease induction. Celecoxib also suppressed the severity of EAE even after the initiation of clinical symptoms. Moreover, celecoxib inhibited EAE in COX-2 deficient mice, indicating that celecoxib inhibited EAE in a COX-2-independent manner. In celecoxib-treated mice, IFN-γ production from MOG-specific T cells was reduced and MOG-specific IgG1 was elevated compared to vehicle-treated mice, suggesting a Th2 bias of MOG reactive T cells in vivo. Infiltration of inflammatory cells was suppressed when treated with celecoxib. These results suggest that celecoxib can be useful as a new additional therapeutic agent for multiple sclerosis. 0S7B-01 Prevention of experimental autoimmune encephalomyelitis via induction of T cells with regulatory function by transfer of ES-DC expressing MOG peptide along with TRAIL Shinya Hirata, Satoru Senju, Hidetake Matsuyoshi, Daiki Fukuma and Yasuharu NishimuraThe Department of Immunogenetics, Graduate School of Medical Sciences, Kumamoto University, JapanPreviously, we reported a strategy to prevent experimental autoimmune encephalomyelitis (EAE) by treatment of mice with genetically modified dendritic cells (DC) presenting myelin oligodendrocyte glycoprotein (MOG) peptide in the context of MHC class II molecules and simultaneously expressing TNF-related apoptosis-inducing ligand (TRAIL) or Programmed Death-1 ligand (PD-L1). For genetic modification of DC, we used a recently established method to generate DC from mouse ES cells in vitro (ES-DC). ES cells were sequentially transfected with an expression vector for TRAIL or PD-L1 and a MOG epitope presenting vector. Subsequently, doubletransfectant ES cell clones were induced to differentiate to ES-DC. In this study, we demonstrated that the severity of myelin basic protein (MBP)induced EAE was also reduced by ES-DC-TRAIL/MOG but not by ES-DC-PDL1/MOG. This preventive effect diminished, if CD4 + CD25 + T cells were depleted by the injection of anti-CD25 mAb into mice before treatment with ES-DC-TRAIL/MOG. In addition, the adoptive transfer of CD4 + CD25 + T cells from ES-DC-TRAIL/MOG-treated mice protected the recipient naïve mice from MOG-or MBP-induced EAE. The number of Foxp3 + cells increased in the spinal cord of mice treated with ES-DC-TRAIL/MOG. These results suggest that prevention of EAE by treatment with ES-DC-TRAIL/MOG is mediated, at least in part, by MOG-reactive CD4 + CD25 + regulatory T cells propagated by ES-DC-TRAIL/MOG. For the treatment of organ specific autoimmune diseases, genetically modified ES-DC presenting some tissue-specific self-antigen along with TRAIL would be effective even when multiple auto-antigens are recognized by autoreactive T cells by the mechanism of epitope spreading. 0S7B-02 Peripheral tolerance induction using ECDI-Fixed APCs for treatment of EAE acts through an indirect mechanisms Ethylcarbodiimide (ECDI) is a chemical crosslinker that couples antigen to cells delivering a tolerizing signal to T cells. In this study we explored the mechanism of ECDI-antigen (Ag)-coupled cell (Ag-SP) as a treatment of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). Ag-SP tolerance has been demonstrated to induce unresponsiveness in autoreactive T cells by presentation of Ag on coupled splenocytes initiating a state of anergy. This tolerance is believed to result from a direct interaction between the host T cell with the fixed donor Agcoupled antigen presenting cell (APC), delivering an Ag specific signal through ligation of the host T cell receptor and peptide:MHC class II complex on the donor APC without costimulation. However, mouse spinal chord homogenate, whole protein, or peptides all function as efficient Ags to induce tolerance suggesting reprocessing of the donor Ag-SP and representation of the dominant epitopes by host APCs. To investigate the mechanism of Ag-SP tolerance we examined donor vs. host MHC class II restriction during Ag-SP tolerance induction. Our data using MHC knockout, allogeneic and/or syngeneic derived donor Ag-SP demonstrate that MHC restriction is not necessary for Ag-SP tolerance induction. It appears that the ECDI-Ag-SP are actively undergoing apoptosis allowing them to function as an Ag carrier to be reprocessed and represented by host APCs. This further supports the promising use of Ag-SP as an antigen specific therapy for autoimmune and other inflammatory disease. Administration of the superagonistic monoclonal anti-CD28 antibody JJ316 is a highly effective treatment in experimental autoimmune encephalomyelitis (EAE) but the underlying mechanism has remained elusive. Using adoptive transfer EAE in the Lewis rat as our model we could now show that JJ316 interferes with the migration of pathogenic T cells to the CNS and that this is responsible for its great potency in preventing the development and progression of EAE. The inhibitory effect is achieved within the secondary lymphoid organs through the antibody's unique ability to expand and activate FoxP3 + regulatory T cells. Importantly, the presence of regulatory T cells within the CNS is dispensable for the beneficial effect of JJ316, irrespective of whether the antibody is administered prophylactically or therapeutically in overt EAE. Still, regulatory T cells preferentially home to the CNS at later time points after disease initiation, suggesting a potentially important role in the resolution phase or in preventing relapses. As to the mechanism at work, we found that administration of JJ316 prevents the disruption of the blood-brain barrier, favours secretion of Th2 cytokines and abrogates IFN-γ production. The latter results in an impaired expression of CXCR-3, the major chemokine receptor of effector T cells, which presumably accounts for the antibody's ability to inhibit the migration of pathogenic T cells to the CNS. This represents the mechanistic basis of a novel therapeutic concept that, we believe, may be translated into clinical practice once humanized monoclonal antibodies with the same specificity become available. 0S7B-04 Oral administration of anti-CD3 antibody suppresses experimental allergic encephalomyelitis by inducing CD4 + CD25 − LAP + regulatory T cells Background: Mucosal administration of auto-and allo-antigens induces immunologic tolerance and is effective in treatment of animal models of autoimmunity, inflammation and transplantation. Parenteral anti-CD3 is efficacious in animal models of autoimmunity and in humans, and anti-CD3 is approved for transplant rejection and positive results have been reported in new onset type 1 diabetes. We investigated the effect of oral anti-CD3 in the animal model of multiple sclerosis, experimental allergic encephalomyelitis (EAE). Methods: We orally administered anti-CD3 or isotype control antibody in doses ranging from 5 μg to 500 μg once per day for 5 consecutive days. Optimal dose was 5 μg; no effect was observed at 50 μg and 500 μg worsened the disease. The PLP-specific immune response showed decreased proliferation, reduced IL-2 secretion and increased IL-10, IL-4 and TGF-β. There was an increase in CD4 + CD25 − LAP + (TGF-β latency associated peptide) T cells with enhanced in vitro regulatory activity that was reversed by anti-TGF-β but not anti-IL-10 and that correlated with the dose which suppressed EAE. Adoptive transfer of mesenteric CD4 T cell but not LAP-depleted CD4 T cells suppressed EAE in a TGF-β dependent fashion. Oral anti-CD3 induces CD4 + CD25 − LAP + regulatory T cells that function in a TGF-β dependent fashion. These results identify a novel and physiologic mechanism to induce regulatory T cells that is clinically applicable to a variety of immune mediated disorders. 0S7B-05 Glatiramer acetate (Copaxone) therapy restores regulatory, cytotoxic, HLA-E-restricted CD8 + T cells in multiple sclerosis One of the goals of successful therapeutic immune modulation of autoimmune disease is the induction of peripheral tolerance, a large part of which is mediated by regulatory/suppressor T cells. We demonstrate here a novel immunomodulatory mechanism by an exogenous peptidebased therapy that incites an HLA Class I-restricted, cytotoxic, suppressor CD8 + T-cell response. We have shown previously that treatment of multiple sclerosis (MS) with glatiramer acetate (GA, Copaxone®) induces differential upregulation of GA-reactive CD8 + Tcell responses. We now show that these GA-induced CD8 + T-cells are regulatory/suppressor in nature. Untreated patients show overall deficit in CD8 + T-cell-mediated suppression, compared to healthy subjects. GA therapy significantly enhances this suppressive ability, which is mediated by cell contact-dependent mechanisms. CD8 + T-cells from GA-treated patients and healthy subjects, but not those from untreated MS patients, exhibit potent, HLA Class I-restricted, GA-specific cytotoxicity. We further show that these GA-induced, cytotoxic CD8 + T-cells can directly kill CD4 + T-cells in a GA-specific manner. Killing is enhanced by preactivation of target CD4 + T-cells and depends on cross-presentation of GA through HLA-E. Thus, we demonstrate that GA therapy induces a suppressor/cytotoxic CD8 + T-cell response, which is capable of modulating in vivo immune responses during ongoing therapy. These studies not only explain several prior observations relating to the mechanism of this drug but also provide important insights into the natural immune interplay underlying this human immune-mediated disease. 0S8A-01 A sensitive non-isotopic assay for acetylcholine receptor autoantibodies Rachel Hewer, Ian Matthews, Shu Chen, Vivienne McGrath, Michele Evans, Emma Roberts, Sarah Nute, Jane Sanders, Jadwiga Furmaniak, Bernard Rees Smith FIRS Laboratories, RSR Ltd., Parc Ty Glas, Llanishen, Cardiff, CF14 5DU, UK Measurement of autoantibodies to the acetylcholine receptor (AChR) is useful in the assessment and management of patients with myasthenia gravis (MG), and development of a new non-isotopic assay for AChRAb which provides specificity and sensitivity at least as high as current isotopic assays is described. In the assay, serum samples (100 μL) were pre-incubated with 25 μL of a mixture of purified foetal and adult-type AChR. AChRAb in 50 μL of the mixture were allowed to compete with three different AChR monoclonal antibodies (MAbs1-3) for binding sites on the AChR: MAb1 was coated onto ELISA plate wells whereas MAb2 and 3 were labelled with biotin and used in liquid phase. MAb-biotin binding was detected by addition of streptavidin peroxidase and substrate. The higher the concentration of AChRAb in the test serum, the greater the inhibition of MAbs binding to the AChR resulting in a reduction in final OD. AChRAb were detected by ELISA in 76/83 MG sera studied (91.6%), whilst 72/83 (86.7%) were positive for AChRAb by immunoprecipitation assay (IPA) based on AChR labelled with 125 I-α bungarotoxin. There were 12/83 discrepant samples; 8 sera were positive by ELISA but negative by IPA (0.33-0.47 nmol/L toxin bound) and 4 sera were negative by ELISA whilst positive by IPA (0.56-2.91 nmol/L toxin bound). All 191 control samples which were negative for AChRAb by IPA, were also negative by ELISA. Overall, results with the AChRAb ELISA and IPA agree well (r = 0.85; n = 83; P < 0.001) and the new assay has good precision and handling characteristics making it suitable for routine use. Antibodies to acetylcholine receptors (AChRs) are present in around 85% of patients with typical generalized myasthenia gravis. A series of experimental approaches has suggested that some of the remaining AChR antibody negative patients have low affinity antibodies to the AChR; these would not be detected by standard immunoprecipitation tests because solubilised AChR is present in monomeric form and at low concentrations (<1 pM). We, therefore, asked whether we could detect antibodies to AChRs by immunofluorescence of cells expressing high density clusters of AChRs. Human embryonic kidney cells (HEK) were transfected with cDNAs for each of the AChR subunits encoding the adult AChR isoform, and co-transfected with GFP-rapsyn. Rapsyn is a cytoskeletal protein that helps to cluster AChRs at the neuromuscular junction. The cells were incubated with patient or control sera and IgG antibodies detected by Texas-red-anti-human IgG. Control sera did not bind appreciably to the AChR clusters. Four positive controls with high AChR antibodies (>1 nM) showed strong IgG binding to the clusters. Ten of ten sera with low AChR antibodies (0.3-1 nM) showed appreciable IgG binding to the clusters. Ten of 18 sera that had been repeatedly negative (<0.3 nM) by standard immunoprecipitation were positive for IgG binding to the clusters These results show that antibodies to AChRs are present in over half the patients previously thought to be AChR antibody negative, and that cell based immunofluorescence assays could prove useful in diagnostic testing. Myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG) are autoimmune disorders in which the acetylcholine receptor (AChR) is the major autoantigen. DNA microarray technology, supported by quantitative real time PCR, immunohistochemistry and flow cytometry, were used to identify new potential drug targets for MG to delineate genes involved in the pathogenesis of the disease.1. The chemokine IP-10 and its receptor CXCR3 were found to be overexpressed in LNC and muscles of EAMG rats and in thymuses and muscles of MG patients. CXCR3 was up-regulated in CD4 + T cells of MG patients. The expression of several phosphodiesterase (PDE) subtypes was upregulated in LNC and muscles of EAMG rats. Pentoxifylline (PTX), a general PDE inhibitor, suppressed the progression of EAMG when treatment started at the acute or chronic stages of disease. Suppression was associated with down-regulation of humoral and cellular AChRspecific responses as well as down-regulation of specific PDE subtypes and up-regulation of Foxp3, a transcription factor essential for CD4 + CD25 + regulatory T cell function. Data mining of the muscle transcriptome revealed two major groups of deregulated genes common to MG and EAMG: (a) Genes linked to muscle biology including muscle proteins regulating contraction such as myosin polypeptides and myosin binding proteins; (b) Genes coding for the chaperone protein category including several heat shock proteins. There were no inflammation-associated deregulated genes in MG or EAMG.Our results demonstrate the power of DNA microarray technology to identify novel genes involved in the pathogenesis of myasthenia and other autoimmune diseases. 0S8A-04 Altered expression of chemokine receptor CXCR5 on T cells of myasthenia gravis patients H. Onodera 1 , R. Saito 2 , H. Tago 2 , Y. Suzuki 2 , Y. Itoyama 2 , Y. Matsumura 2 , T. Kondo 2 Myasthenia gravis (MG) is characterized by the T cell-dependent production of anti-acetylcholine receptor antibodies. The chemokine receptor CXCR5 regulates lymphocyte migration and is expressed specifically on a subset of CD4 + T cells named follicular helper T cells (TFH), the key modulators of antibody production by B cells. We studied the frequency of CXCR5-positive lymphocytes in the peripheral blood of MG patients before and after therapy Untreated MG patients showed a significantly higher frequency of CXCR5 + CD4 + T cells in the peripheral blood compared with the control group, while no significant difference in the percentages of CXCR5 + CD4 + T cells was observed between the patients of the hyperplasia group and those of the thymoma group. The CXCR5 + CD4 + T cell frequency correlated with the disease severity. The CXCR5 + CD4 + T cell frequency of MG patients positive for other autoantibodies together with anti-AChR antibodies was significantly higher than in those having only anti-AChR antibodies. After therapy, the CXCR5 + CD4 + T cell percentage decreased gradually to the control level with a significant inverse correlation between the CXCR5 + CD4 + T cell frequency and duration after the initiation of MG therapy. These results suggest that CXCR5 + CD4 + T cells play an important role in the disease activity of MG and that some MG patients have a systemic abnormality in T cell-dependent antibody production. 0S8A-05 Childhood myasthenia gravis in Japan Y. Nomura, K. Hachimori, Y. Nagao, M. Segawa, K. Kimura and M. Segawa Segawa Neurological Clinic for Children, Tokyo, JapanObjective: The ages at onset of myasthenia gravis (MG) in Japan show two modal patterns, the highest under 3 years of age, which is associated with HLA DR9/13-DQ6/9, followed by young adult peak. Clinical characteristics, treatments and long term prognosis of the childhood MG are presented.Materials and methods: 270 cases of childhood onset MG who visited this clinic in the past 32 years were evaluated. Clinical types were divided to ocular, latent general (clinically only ocular symptoms, but electrophysiologically myasthenic in the extremity muscles) and general. Treatment consisted with anticholinesterase, steroid, thymetomy or tacrolimus.Results: Clinical types showed general 30%, latent general 50% and ocular 20%. Acetylcholine receptor antibody (AChR Ab) are negative or low in most cases, but some cases with minimum ocular symptoms as sequelae begin to show elevation of AChR Ab at around 20 years of age. 60% of cases responded to steroid and less than 10% of cases needed thymectomy. Steroid showed best effects when started within 3 years from onset. Onset older than 11 years and general type often needed thymectomy. Tacrolimus was used in 10 cases with good results.Conclusion: Specific immature genetic background seems to play important roles in Japanese childhood MG. Long term prognosis of childhood MG is generally good, but early intervention by steroid or thymectomy is important for the complete remission. Tacrolimus is safely used to switch from steroid. 0S8B-01 The effect of statin on experimental autoimmune neuritis T. Kiyozuka, T. Fujioka, T. Kudeken, K. Murata, H. Ito, and Y. Iwasaki Toho University Omori Hospital, Tokyo, JapanDisease-modulating effect of statin on multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) was unveiled. However, its effect on autoimmune disorders of peripheral nervous system (PNS) remains unclear. To investigate the effect of statin on experimental autoimmune neuritis (EAN), atorvastatin was given orally to EAN rats daily during an entire course. EAN rats fed with vehicle only were used as control. Clinical signs were assessed daily using 8 gradescore system. Cauda equina (CE) were removed at 4, 10, 12, 14, 18, 21, and 24 days post-immunization (DPI). mRNA expression of IFN-gamma and IL-10 in the CE was studied using semi-quantitative RT-PCR. Both groups developed EAN at 12 DPI simultaneously although atorvastatin-treated rats showed milder signs and earlier recovery. Mononuclear cell infiltration seen in the CE of atorvastatin-treated rats was also milder than that of control rats. RT-PCR revealed that IFN-gamma mRNA expression at 10 DPI and IL-10 mRNA expression at 12 DPI in atorvastatin-treated rats were decreased compared to the control rats. In EAE, atorvastatin causes shift from Th1 to Th2 cytokine balance resulting in suppression of the neurological deficit. Out data showed that, in EAN, atorvastatin only suppressed Th1 cytokine irrelevant to Th2 cytokine up-regulation as seen in EAE. This difference may reflect different, yet unknown mechanism of modulation of autoimmune inflammation between CNS and PNS. 0S8B-02 Double-stranded RNA induces inflammatory gene expression in Schwann cells, sensory neuronal death, and peripheral nerve demyelination Inflammation in the peripheral nervous system (PNS) is one of the characteristics of virus-induced peripheral neuropathy. In this inflammatory response, Schwann cells are actively involved. Recently, toll-like receptor 3 (TLR3) was identified as a receptor for double-stranded RNA (dsRNA) that induces anti-viral and inflammatory responses in cells of the innate immune system. In this study, we investigated the expression of TLR3 and the TLR3-mediated signaling pathways in Schwann cells. TLR3 was constitutively expressed in the primary rat Schwann cells and iSC, an immortalized Schwann cell line. Stimulation with poly(I:C), a synthetic dsRNA, induced the expression of various inflammatory genes such as IP-10, RANTES, TNF-α, and iNOS in Schwann cells. Studies on the intracellular signal transduction pathways using iSC revealed that dsRNA induces the activation of NF-κB, p38, and c-Jun N-terminal kinase (JNK). The activation of NF-κB, p38, JNK, and PKR is required for dsRNA-mediated iNOS gene expression. However, the activation of PI3 kinase and GSK-3β inhibited iNOS gene induction, a process mediated by their inhibitory effects on NF-κB and p38 activation. NO production induced by dsRNA caused neuronal cell death in cultured dorsal root ganglion (DRG). Finally, the introduction of dsRNA into the rat sciatic nerve induced iNOS gene expression and peripheral nerve degeneration in vivo. Taken together, these data suggest that viral RNA may induce inflammatory Schwann cell activation and peripheral nerve damage in the PNS. 0S8B-03 Anti-ganglioside complex antibodies in Miller Fisher syndrome We recently reported that some ganglioside complexes (GSCs) are target antigens for serum antibodies in patients with Guillain-Barré syndrome (GBS) and that anti-GSC antibodies may be associated with particular clinical features of GBS. Miller Fisher syndrome (MFS), characterized by a clinical triad of ophthalmoplegia, ataxia and areflexia and by the presence of the serum IgG anti-GQ1b antibody, is considered to be a variant of GBS. The purpose of this study is to investigate antibodies to GSCs in sera of patients with MFS. We investigated IgG antibodies to GSCs in sera of 12 consecutive MFS patients using ELISA. Test ganglioside antigens were GM1, GM2, GD1a, GD1b, GT1a, GT1b, GQ1b, and GSCs containing two of the above seven antigens. There were at least three different specificities in MFS-associated antibodies; GQ1b-specific, anti-GQ1b/GM1-positive, and anti-GQ1b/GD1a-positive. Not only GQ1b and GT1a themselves but also clustered epitopes of GSCs including GQ1b or GT1a may be candidates as prime target antigens for serum antibodies in MFS patients. 0S8B-04 Complex formation of bacterial GM1-and GD1a-like lipo-oligosaccharides can make GM1b mimicry for autoantibody production Campylobacter jejuni strains with class A lipo-oligosaccharide (LOS) biosynthesis locus exceptionally carry both GM1-and GD1a-like LOSs and have been found worldwide to be associated with Guillain-Barré syndrome (GBS) development. We assumed that complex formation of these ganglioside epitopes makes the organisms express new structure, as a result generating autoantibody which has different reactivity from antibodies specific for each ganglioside. In this study, we detected IgG autoantibody which specifically reacts with mixture of GM1 and GD1a, not with each ganglioside, in several GBS patients from whom C. jejuni strains carrying both GM1-and GD1a-like LOSs had been isolated. All of these patients had higher titer of anti-GM1b IgG antibody, and absorption study showed that anti-GM1b and anti-GM1/GD1a complex antibodies were absorbed by GM1/GD1a-like LOS. Furthermore, these absorptions were lost when we used LOS as absorber after treatment with neuraminidase from Arthrobacter ureafaciens, which converts GM1/GD1a-like structure to GM1-like one.Immunization with a class A strain (CF90-26; GBS-related), which carries both GM1-and GD1a-like LOSs, induced IgG antibody response against GM1b as well as GM1/GD1a complex, but not individual GM1 or GD1a. These findings strongly suggest that target structure for anti-GM1/GD1a complex antibody mimics GM1b ganglioside, which has structurally different sugar sequence from GM1 and GD1a, but could also be directed for as target antigen for autoantibody in GBS. This is the first example demonstrating that complex formation of bacterial structures is another way to make molecular mimicry in autoantibody production. 0S8B-05 Efficacy of ciclosporin in chronic inflammatory demyelinating polyneuropathy patients who require repeated treatment with intravenous immunoglobulins Background: Primary goals of current therapies used for chronic inflammatory demyelinating polyneuropathy (CIDP) are to control symptoms, improve functional ability, and maintain long-term remission. Some CIDP patients whom corticosteroids cannot sustain remission, require repeated treatment with intravenous immunoglobulins (IVIG). Objective: To investigate the therapeutic efficacy of ciclosporin for CIDP patients who require repeated IVIG, and to evaluate basic information and target samples size for therapeutic trial with ciclosporin. Methods: Ciclosporin was tried for 14 CIDP patients whom corticosteroids could not sustain remission. An initial dose of ciclosporin was 3 mg/kg/day with plasma trough concentrations between 100 and 150 ng/ml. Frequency of symptomatic relapses and side effects of 11 patients treated with ciclosporin for 12 months were investigated. Results: Eight patients responded to ciclosporin and sustained remission over 12 months; 2 gradually prolonged remission interval after increase of ciclosporin dose; 1 showed no improvement and repeated IVIG were required. Except for 1 patient with renal function disturbance, there have been no serious side effects. Conclusions: Ciclosporin is effective therapeutic agent for long-term remission in patients with CIDP who do not show sustained improvement under corticosteroid therapy. Randomized parallel group comparison multicenter collaborative research, attending hospitals throughout Japan, will start soon. The current study used trimethyltin-induced damage to examine the contribution of tumor necrosis factor (TNF) superfamily in hippocampal dentate granule cell (DG) death and to determine if differential expression of the TNF receptors and relationship to glia would characterize neuronal vulnerability. In the DG, TNFα, TNFR1, TNFR2 mRNA levels (QT-PCR of laser captured material) were elevated; microglia activated; astrocytes hypertrophic. In the protected CA pyramidal layer, TNFα, TNFR1, TNFR2, mRNA levels were elevated; astrocytic processes thickened, and reactive non-phagocytic microglia present. Microglia proliferation was 11, 20, and <1% in the DG, CA, and CA1, respectively. With injury, intracellular distribution of TNFR1 protein in the DG shifted from membrane to cytoplasmic/nuclear, cells expressed active caspase 3 and were in contact with activated microglia. In the CA, intracellular distribution of TNFR2 shifted and cells maintained close contact with GFAP + astrocytes. Neutralizing antibody to TNFα was neuroprotective; mice null for both TNFR1 and 2 showed damage attenuation; mice null for either TNFR1 or R2 showed exacerbation. Basal MIP-1α mRNA levels were elevated over wildtype; with similar induction. Higher basal levels and induction of TNFα mRNA occurred in the TNFR1 −/− and the elevation in both TNFα and IL1α in the TNFR2 −/− mice occurred at the lower dose. These results suggest a sequence of events: proinflammatory cytokines, activation of death receptors, and microglia phenotype determining pattern of neuronal death. Objective: Our goal was to determine whether the EP1 prostanoid receptor contributed to the excitotoxic death of neurons. Methods: EP1 receptor antagonists (Ono 8711 and SC51089) were examined for neuroprotective effects against an excitotoxic exposure to nmethyl-d-aspartate (NMDA). This was tested in two preparations of primary neuronal cultures containing greater than 90% neurons (PN) or a mixture of neurons and glia. Results: Treatments of Ono 8711 (3, 10 or 30 nM), SC51089 (10 μM) or the COX-2 inhibitor NS398 (30 μM), increased the number of neurons that survived NMDA by a factor of three in PN cultures. The percent of neurons that expressed EP1 was 65% in mixed cultures and 90% in PN cultures. NMDA induced a ten-fold increase in PGE2 in both culture preparations, although the net amount of PGE2 was about five-fold higher in the mixed cultures. Combined, these findings indicate that a lack of neuroprotection in mixed cultures by EP1 antagonists was not due to a lack of neuronal EP1 expression nor was it due to an inability to synthesize PGE2.Conclusion: These findings indicate that activation of EP1 on neurons contributes to neuronal death and can be influenced by non-neuronal cells. PP01-03 Thioredoxin inhibits NMDA-induced neurotoxicity in the rat retina Thioredoxin (TRX), an endogenous redox-activeregulator, plays a cytoprotective role against focal ischemic, excitotoxic, and 1-methyl-4phenylpyridinium-induced brain damage. We investigate whether TRX modulates N-methyl-D-aspartate (NMDA)-induced retinal neurotoxicity. TRX, mutant TRX, or saline were injected together with NMDA intravitreously into rat eyes. Number of retinal ganglion cells (RGCs) was counted by retrograde labeling analysis to evaluate retinal damage. Cellular apoptosis in the retina was measured by TdT-mediated dUTP nick-end labelling (TUNEL) and by measurements of caspase activities. Activation of the mitogen-activated protein kinases (MAPKs), p38 and JNK, and MAPK kinases (MKKs), MKK3/6 and MKK4 were examined by Western blot and immunohistochemistry. Protein carbonylation, nitrosylation and lipid peroxidation were assessed to evaluate antioxidative effects of TRX. RGCs were rescued by TRX, compared with saline, when evaluated by retrograde labelling analysis, 7 days after NMDA-injection. The induction of caspase-3 and caspase-9, but not caspase-8, by NMDA was significantly lower in TRX-treated eyes than in saline-treated eyes. NMDA-induced activation of p38 and JNK after 6 h, and of MKK3/6 and MKK4 after 3 h, was markedly suppressed in RGCs by TRX, but not by the mutant form. NMDA-induced increases in protein carbonylation, nitrosylation, and lipid peroxidation were also suppressed in TRX-treated eyes. TRX effectively attenuates NMDA-induced retinal cell damage, and suppression of oxidative stress and inhibition of apoptotic signalling pathways are involved in this cytoprotective effect. PP01-04 Trail-induced death of adult human oligodendrocytes is mediated by JNK and mitochondrial activation Background: TRAIL (TNF-related apoptosis inducing ligand) is a member of TNF family that induces death of mainly tumor cells. TRAIL is expressed on autoreactive T cells and induces death of adult human oligodendrocytes by ligation of TRAIL-receptor 1. Intracellular signaling involved in oligodendrocytes injury is still unknown. Methods: In our study we investigated involvement of JNK and mitochondrial pathway in death after TRAIL stimulation. JNK activation was assessed by using antibodies against phosphorylated JNK isoforms and analyzing c-jun phosphorylation by autoradiography. Changes in mitochondrial membrane permeability was assessed using ApoAlert Mitochondrial Membrane kit. Cell death was analyzed by FACS using Annexin-V and PI staining. Results: Our results shown that intracellular signaling involved in TRAILinduced death of oligodendrocytes is associated with strong, sustained activation of JNK isoform 3. After TRAIL stimulation was observed changes in mitochondrial membrane potential. Conclusions: These results indicate that JNK and mitochondrial activation are critically involved in oligodendrocytes death induced by TRAIL and might have importance in protection immune-mediated oligodendrocytes demise. Tumor necrosis factor (TNF) induces apoptotic-like cell death of oligodendrocytes, the cell type targeted in MS. The ligation of TNF receptors induces several signal transduction pathways including caspase cascade and mitochondrial derived factors like apoptosis inducing factor (AIF). However, the precise mechanism involved in human oligodendrocyte (hOL) death, is unknown. OLs were prepared from human brain specimen. AIF activity was assessed by hOLs DNA electrophoresis and its nuclei translocation with confocal microscopy. Caspase inhibitor, ZVAD.FMK, calpain inhibitor, ZLLY.FMK, serine proteases inhibitor, TPCK, and cathepsin inhibitors, ZFL and ca-074-Me, were used to assessed their involvement in OL death. TNF-induced death of hOLs is noncaspase dependent, as evidenced by: lack of generation of caspase-8, -1 and -3 active subunits; lack of cleavage of caspase-1 and -3 fluorogenic substrates; and lack of hOL death inhibition by ZVAD.FMK. Also calpain, serine proteases, and cathepsin inhibitors were inefficient in prevention of hOLs death. In contrary, TNF-induced hOLs death involved AIF as evidenced by characteristic for AIF-induced cell death large scale DNA fragmentation and AIF translocation to hOLs nuclei upon TNF exposure. These results indicate that TNF-induced death of hOLs depends on AIF, information of significance for the design strategies to protect hOLs during immune-mediated demyelination. PP01-06 C5b-9 complement complex protects oligodendrocytes from apoptotic cell death by inhibiting caspase-8 processing and upregulating FLIP Activation of the terminal complement cascade involving C5 to C9 proteins has a beneficial role for oligodendrocytes in experimental allergic encephalomyelitis, an animal model of multiple sclerosis, by protecting them from apoptotic cell death. We have previously shown that sublytic C5b-9 complexes, through posttranslational regulation of Bad, inhibit the mitochondrial pathway of apoptosis induced by serum deprivation. In serum-free defined medium oligodendrocytes undergo apoptosis and differentiation concomitantly. Under this condition, we found that caspase-8 processing was increased in association with Bid cleavage and markedly reduced expression of c-FLIP L protein. The caspase-8 inhibitor Z-IETD-FMK inhibited cell death associated with differentiation in a dosedependent manner. Phosphatidylinositol 3-kinase inhibitor LY294002 reversed these C5b-9 effects. These data suggest that C5b-9 through phosphatidylinositol 3-kinase signaling can rescue OLG from Fas-mediated apoptosis by regulating caspase-8 processing. PP01-07 MEK-ERK signaling is involved in interferon-γ-induced death of oligodendroglial progenitor cells T. Itoh, A. Itoh and M. Horiuchi Department of Neurology, University of California Davis/Institute for Pediatric Regenerative Medicine, Shriners Hospital for Children Northern California, Sacramento, California, USA Oligodendrocytes are exposed to various cytokines in inflammatory lesions in the CNS. In this study, we focused on the direct effects of interferon-gamma (IFNG) on purified rat oligodendroglial cultures at different developmental stages. Among the three stages tested, IFNG had direct cytotoxic effects on actively proliferating oligodendrocyte progenitors, but much less on immature oligodendrocytes and none on mature oligodendrocytes. This stage specific susceptibility of progenitors to IFNGinduced cytotoxicity consisted of two components, delay in the cell cycle transition from the G1 phase to the S phase and increased cell death at least partly mediated by apoptosis, suggesting that progression of the cell cycle was tightly linked to this toxic mechanism. As far as could be determined by examining induction of IRF1 mRNA in response to IFNG, there was no functional difference in the signal transducers and activators of transcription (STAT) pathways between progenitors and mature oligodendrocytes. We found that partial inhibition of the ERK pathway, one of mitogen activated protein kinase pathways, by U0126 partially reversed the IFNG-induced cytotoxicity. In addition, ERK activity was quickly downregulated after differentiation of progenitors to immature oligodendrocytes. Therefore, we concluded that simultaneous activation of the STAT pathway by IFNG and the ERK pathway by trophic factors played a role in the stage specific IFNGinduced cytotoxicity in oligodendroglial progenitors. Activation of Akt attenuates apoptosis mediated through both the extrinsic death receptors and via the intrinsic mitochondrial pathway. A highly conserved protein, SAMSN1, predicted to contain 373 amino acids and three Akt consensus phosphorylation sites was identified. The SAMSN1mRNA arises by alternative splicing of a 10 kb gene on chr.21q11 also encoding the recently described HACS1 protein. SAMSN1 mRNA was predominantly expressed in tissues of hematopoietic origin and was detected in mature B and T lymphocytes and macrophages but not in epithelial lines or primary fibroblasts. Co-expression of SAMSN1 with Akt lead to PI-3 kinase dependent, phosphorylation of SAMSN1 and its interaction with Akt. To explore the cellular localization and function of SAMSN1, wild type SAMSN1and mutants lacking either the Akt phosphorylation sites (Tri-A mutant), nuclear localization signals (Δ-NLS), or SAM domain (Δ-SAM) were expressed in 293 T cells. SAMSN1, as well as the Tri-A and Δ-SAM mutants, but not the Δ-NLS mutant, localized to the nucleus indicating that Akt phosphorylation did not regulate cell localization. Jurkat T cells transduced with a retroviral vector expressing WT SAMSN1 were significantly protected from Fas, but not staurosporin, mediated apoptosis and this protection required the putative Akt phosphorylation sites. Together, these data suggest that SAMSN1 as a novel Akt substrate localized to the nucleus of cells of hematopoietic origin that mediates protection from Fas mediated apoptosis. PP01-09 The role of adenosine receptors in IL-6-dependent neuroprotection The immunological response in the brain plays a crucial role in neuropathological conditions. Nowadays, it is consensual that several inflammatory mediators, such as the immunoregulatory cytokine interleukin-6 (IL-6), protect neurons from glutamate-induced neurotoxicity in vitro and in vivo. Despite a large body of experimental evidence, the fundamental mechanisms underlying IL-6-mediated neuroprotection are not understood until today. Our data clearly show that IL-6 is not a neuroprotective agent per se, but that IL-6 instead enhances the expression and function of neuronal adenosine A 1 receptor in vitro and in vivo. Accordingly, we provide evidence that IL-6 by enhancing neuronal adenosine A 1 receptor function (1) efficiently protects hippocampal neurons from hypoxia in acutely prepared brain slices; (2) facilitates neuronal survival in an excitotoxicity paradigm; and (3) protects animals from chemically induced convulsing seizures. It is moreover shown that IL-6-dependent neuroprotection is abolished when the function of neuronal adenosine A 1 receptors is blocked. Based on this data we suggested that IL-6 increases neuronal survival in vitro and in vivo by enhancing the function of neuronal adenosine A 1 receptors, the brains major protective system. PP01-10 Interferon β counteracts the apoptotic cell death induced by TNF α in primary astrocytes through stimulation of PI-3K and MEK Although interferon β (INFβ) has been demonstrated to be effective in the treatment of multiple sclerosis (MS), the mechanism(s) underlying its beneficial effects has not been uncovered. In the present work we have investigated the ability of INFβ to counteract the apoptosis induced by TNFα treatment in primary astrocytes.Primary monolayer cultures of rat cortical astrocytes (E15) were treated with different doses of TNFα (1, 5, 25, 50 or 100 ng/mL) for 24 h in the absence or the presence of INFβ (2 ng/mL). Apoptotic cell death was determined by Hoechst 33258 staining. Phosphorylated Akt and ERK were determined by Western blot.TNFα treatment induced a significant increase in astrocyte death starting at the dose of 25 ng/mL. This effect was blocked in the presence of the caspase-8 inhibitor Z-IETD-FMK, indicating that astrocyte death is due to apoptosis. Co-treatment of cultured astrocytes with INFβ significantly reduced TNFα-induced cell death. This antiapoptotic effect of INFβ depends, at least partially, on its ability to stimulate the phosphorylation of Akt. When PI-3K activity was inhibited by means of LY294002 treatment, INFβ failed to promote both Akt phosphorylation and astrocyte survival. In addition INFβ also induced phosphorylation of ERK. Inhibition of MEK activity by means of PD98059 treatment, blocked the ability of INFβ to counteract TNFα-induced apoptosis.On the view of these results it is tempting to speculate that the beneficial effects of interferon treatment on MS patients may depend on its ability to prevent astrocyte death.Supported by FISS grant no. PP02-01 TLR3 triggers an anti-viral response in astrocytes Toll-like receptors (TLRs) are receptors that mediate innate immunity against pathogens. Toll-like receptor 3 (TLR3) is a viral nucleic acid sentinel activated by dsRNA within intracellular vesicles. TLR3 signals through TRIF (TIR domain-containing adaptor-inducing IFNβ) and TRAF6 to activate interferon regulatory factor 3 leading to the production of IFNβ (primary response), and to a delayed activation of NF-kB leading to the production of proinflammatory cytokines and chemokines. To explore the spectrum of genes induced in human astrocytes by TLR3 we used a microarray approach and the dsRNA mimetic poly I:C (pIC) as ligand. One of the highly induced genes was viperin/cig5, a protein with known anti-viral activity. Viperin/cig5 expression was dependent on IRF3 and NF-kB signaling, and repetitive stimulation with poly(I:C) but not IL-1 further increased expression. To determine whether poly(I:C) induced an antiviral state in astrocytes, a pseudotyped HIV viral particle VSVg-env-HIV-1 was used. Poly(I:C) significantly abrogated HIV-1 replication whereas IL-1, which also potently activates astrocytes, did not. Viperin induction could also be substantially inhibited by neutralizing antibodies to IFNb, as could HIV-1 replication. To explore a role for viperin in IFNbmediated inhibition of HIV-1 we employed an RNAi approach. RNAi directed against viperin, but not a scrambled RNAi, significantly inhibited viperin expression, and also significantly reversed poly(I:C)-induced inhibition of HIV-1 replication. We conclude that viperin contributes to the antiviral state induced by TLR3 ligation in astrocytes, supporting a role for astrocytes as part of the innate immune response against infection in the CNS. PP02-02 Toll-like receptor profiles and functions in the human brain during multiple sclerosis Toll-like receptors (TLRs) are key elements in the control of inflammatory pathways also in the human CNS. Marked differences, however, separate TLRs on either type of glial cell. In microglia, a rather diverse set of TLRs are expressed and at least TLR3 and TLR4 can be found only inside endosomal vesicles. Astrocytes on the other hand, express a more limited set of TLRs, and only on their cell surface.In our present studies we have found that upon activation astrocytes display a striking preference for expressing elevated levels of TLR3 only. Genomic profiling and functional data indicate that TLR3 activation on astrocytes does not activate a traditional pro-inflammatory response but, instead, a comprehensive neuroprotective, angiogenic and anti-inflammatory response. To shed further light on TLR functions in the human CNS during inflammation, TLR profiles in human brain samples including multiple sclerosis lesions of varying stages were evaluated by real-time PCR and protein staining techniques. Rochford 1 and Harald Neumann 1,3 1 Neuroimmunology Unit, European Neuroscience Institute, Goettingen, Germany; 2 Department of Neurology and Neurobiology of Aging, Kanazawa University Graduate School of Medical Science, Japan, and 3 Institute of Reconstructive Neurobiology, University Bonn LIFE and BRAIN Center, Bonn, GermanyBackground: Elimination of apoptotic neurons without inflammation is crucial for brain tissue homeostasis, but the molecular mechanism has not been firmly established. Triggering receptor expressed on myeloid cells-2 (TREM2) is a recently identified innate immune receptor. We analyzed microglial function in response to TREM2 stimulation or after knock down of TREM2. Methods: Cultured microglia were transduced with Flag-tagged TREM2, short hairpin TREM2 for RNA interference, wild type TREM2 for over-expression, or their control GFP lentiviral vectors. Function in response to TREM2 stimulation, after knock down of TREM2 and co-culture with apoptotic neurons was analyzed by flow cytometry, RT-PCR, and immuno-cytochemistry. Results: TREM2 stimulation increased phagocytic activity, but neither induced inflammatory mediators nor up-regulated surface markers were involved in antigen presentation. Furthermore, TREM2 knock down microglia showed impaired clearance of apoptotic neurons and increased gene transcription of TNFalpha, IL-1beta and NOS2. Over-expression of TREM2 in microglia resulted in increased phagocytosis of apoptotic neurons and decreased gene transcription of inflammatory mediators. Conclusion: TREM2 signaling participates in clearance of apoptotic neurons and down-regulation of inflammation to maintain the immunoprivileged CNS microenvironment. PP02-04 Primate microglia are particularly sensitive to TLR8-mediated activation but do not react to TLR7 and TLR9-mediated activation Microglia are CNS resident innate immune cells that can become activated upon encountering infectious agents. Toll-like receptors (TLR) are a diverse family of receptors recognizing pathogen-associated molecular patterns that drive activation of the expressing cell. The transformation of resting microglia into activated APC involves multiple steps and the outcome will be determined by the differentiation potential of resting microglia and by the activating stimulus. In order to study this process, we used primary microglia obtained from adult rhesus monkeys. After exposure to different differentiation (M-CSF or GM-CSF) regimes, functional reactivity to TLR-mediated signaling was determined and TLR mRNA expression levels were quantified.Both types of microglia responded to TLR2, 3, 4, 5 and 8-mediated stimulation as measured by the production of TNF-alpha, IL-6, IL-12 and IL-23. Equally important, rhesus microglia were not responsive to TLR7 and 9mediated signaling, this latter in contrast to murine microglia. Both types of microglia were particularly responsive to TLR8-mediated stimulation. However, TLR8-mediated activation evoked a much stronger cytokine response by M-CSF-differentiated microglia as compared to GM-CSF-differentiated microglia, whereas reactions towards other TLR ligands were comparable. Moreover, M-CSF-differentiated microglia expressed higher TLR8 mRNA levels than GM-CSF-differentiated microglia. Since TLR8-mediated signaling activates microglia so potently, we are currently screening CNS material of different origin for the presence ofendogenous -TLR8 ligands. Intriguingly, TLR-mediated activation of microglia dramatically enhanced TLR9 mRNA expression levels in GM-CSF-differentiated microglia only, possibly endowing these cells with sensitivity to TLR9-mediated signaling. PP02-05 Release of activin A by microglial cells upon stimulation with bacterial TLR-agonists S. Ebert, R. Nau, U. MichelGeorg-August-University, Department of Neurology, Göttingen, GermanyObjective: Activin A, a member of the transforming growth factor-beta family of growth and differentiation factors, is a multifunctional cytokine, with one of its roles being in the immune system and inflammatory processes. It has neuroprotective properties and can be detected within the central nervous system. We previously demonstrated that concentrations of activin A are elevated in the cerebrospinal fluid (CSF) of patients with meningitis. As the sources of elevated activin A concentrations in CSF during meningitis have not yet been discovered, we examined whether microglial cells release activin A upon stimulation with bacterial Toll-likereceptor (TLR)-agonists. Methods: Primary mouse microglial cells were exposed to agonists of TLR2 [Pam3Cys (Tripalmitoyl-S-glyceryl-cysteine)], TLR4 [LPS (lipopolysaccharide)], and TLR9 [CpG (oligonucleotides containing unmethylated cytosinguanosin motifs)] for 24 h. Activin A concentrations in the cell culture supernatants were measured by ELISA [median (minimum / maximum)]. Results: While activin A concentrations in the cell culture supernatants were below the detection limit of the ELISA in the control group, they were significantly elevated after treatment with the different TLR-agonists: 279 pg/ ml (196/337) after 1μg/ml LPS (p < 0.0001), 117 pg/ml (0/167) after 1 μg/ml Pam3Cys (p = 0.007), and 77 pg/ml (0/185) after 10 μg/ml CpG (p =0.02). Discussion: Our results show for the first time that microglial cells release activin A upon activation by bacterial TLR-agonists. This finding provides further evidence for a role of activin A in the innate immune response and suggests that microglial cells are a source of elevated activin A concentrations observed in the CSF during bacterial meningitis. In this study, we describe that LPS is a strong inducer of SOCS-3 expression in macrophages and microglia, which occurs at the transcriptional level. An analysis of the SOCS-3 promoter indicates that AP-1 and two GAS elements are involved in LPS-induced SOCS-3 transcription in macrophages. Furthermore, LPS induces activation of the mitogen-activated protein kinase (MAPK) pathway, which is also involved in LPS-induced SOCS-3 gene expression. We propose that LPS-induced SOCS-3 expression results in a negative feedback loop to attenuate LPS and cytokine-induced immune and inflammatory responses in the CNS. PP02-08 Heterogeneous origin of reactive microglia in injured rat and mouse central nervous system Microglia serve homeostatic functions in normal CNS, and sense threats to and modulate neuronal survival in injured and diseased CNS. Presently, our understanding of microglial biology and involvement in disease is influenced by observations that exogenous bone marrow (BM)derived cells contribute to reactive microgliosis in the mouse. To determine whether there are species-differences in the recruitment and microglial transformation of BM-derived cells into injured CNS, we analyzed the microglial reaction in radiation BM chimeric mice and rats subjected to classical forms of experimental manipulation, known to elicit predictable, strong microglial reactions with no or minimal involvement of monocytic macrophages. We found that a subpopulation of the reactive, bushy-type microglial cells in the dentate gyrus originated from transformed BM-derived cells after entorhinal cortex lesion in BM chimeric mice. This contrasted BM chimeric rats subjected to global cerebral ischemia, which displayed the characteristic microglial rod cell transformation and microglial hyperplasia in regio superior hippocampus, but in the absence of recruitment and microglial transformation of BM-derived cells. Our results provide evidence that reactive microglia in the rat originate from resident microglia, while having mixed BM-derived and resident origin in the mouse. The data emphasize that there are important species-differences in recruitment of blood-borne microglial precursors to the brain and that speciesdifferences may be an issue when considering using BM-derived cells for gene delivery to the CNS in human neurological disorders. Importantly, BM chimeric rats may provide valuable models to establish conditions for how to attract genetically manipulated BM-derived cells into human CNS. PP02-09 Microarray analysis of microglia versus bone marrow-derived macrophages Vuaillat-savarin Carine 1 , Wierinckx Anne 2 , Rey Catherine 3 , Lachuer Joël 1,2 , Belin Marie-Françoise 1 , Nataf Serge 1 However, the molecular basis for such a specialization is poorly known. In particular, markers which could reliably discriminate microglia from other macrophages, have not been yet identified. In the present work, we used gene array analysis to compare the RNA profiles obtained from microglia versus bone-marrow derived macrophages (BMM). Microglial cells isolated from mixed glial cell cultures were driven toward a "resting" state by growing them for 4 days in the presence of a glial cell conditioned medium. In parallel experiments, BMM were generated by stimulating bone-marrow derived myeloid progenitors with M-CSF, for 4 days. After a control of RNA quality (BioAnalyzer 2100, Agilent), double amplification process was performed before cDNA synthesis. The expression of 20,000 genes was then assessed on a CodeLink plateform from Amersham and data were analyzed with Genespring software from Silicon Genetics. To validate our results, genes of interest were selected and their relative level of expression was evaluated by real-time RT-PCR. A number of immune-related genes were identified including genes coding for MHC class II molecules (up-regulated in macrophages), the IL-10 receptor (up-regulated in microglia,) or the erythropoietin receptor (up-regulated in microglia). However, we also identified genes that were not related to immune functions. In particular, several genes involved in lipid metabolism were found to be specifically expressed by microglia. PP02-10 Characterization of the phagocytic activity of mouse primary microglia towards apoptotic glioma cells Microglia ingest various pathogens as well as apoptotic T cells or neurons. This potent phagocytic capacity has hardly been analysed in the context of brain tumour where it could serve the purpose of removing dying but also living tumour cells, as described for macrophages. We herein report on quantitative studies, based on flow cytometry assays, and qualitative studies, using time-lapse video microscopy, that we conducted with primary cultures of microglia derived from three mouse strains and three different glioma cell lines. The phagocytic capacity of microglia was evaluated by determining their phagocytic rate (percent of microglia from a total population that phagocytosed tumour cells within a given time). Microglia from either BALB/c, C57BL/6 and VM/Dk strains all phagocytosed etoposide-treated, apoptotic glioma cells. The phagocytic rate varied between 30% and 70% according to the strain but not to the glioma cell line used. No significant uptake of living tumour cells was observed. Beside the known induction of microglial toxic activity, LPS and interferon-gamma stimulation increased the phagocytic rate, save for VM/Dk-derived microglia. As expected, amoeboid microglia present in the unstimulated population were the only phagocytes, whereas upon stimulation this activity was also observed for rod-shaped cells. Immune stimulation enhanced motility and exploratory behaviour. Stimulated microglia not only killed tumour cells but also phagocytosed dead cells present in their close vicinity. Altogether our data indicate that microglia can phagocytose apoptotic but not living tumour cells. Current work focus on the identification of receptors and ligands mediating microglia-glioma cell recognition. The pathogenesis of many neurodegenerative disorders is exacerbated by an imbalance between matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). We previously reported differential TIMP-1 expression in acute versus chronic astrocyte activation, and in brain tissue of patients with HIV-1-associated dementia (HAD). We now investigate possible mechanisms underlying chronic TIMP-1 downregulation in neuroinflammation. We used interleukin (IL)-1β as a model proinflammatory stimulus and measured mRNA stability in U87 astroglioma cells and transcriptional regulation in activated astrocytes. The half-life of TIMP-1 mRNA was quantified using real time PCR to determine the effects of IL-1β stimulation on the stability of TIMP-1 transcripts. TIMP-1 promoter activation was measured using TIMP-1-luciferase reporter constructs in transfected astrocytes. Our results demonstrate that enhanced TIMP-1 mRNA stability may lead to increased TIMP-1 levels following acute activation. However, chronic downregulation of astrocyte-TIMP-1 is likely through promoter regulation and loss of mRNA stabilization. Furthermore, other proinflammatory cytokines including tumor necrosis factor (TNF)-α and interferon (IFN)-γ, along with HIV-1, enhance the effects of IL-1β on astrocyte-TIMP-1 promoter regulation. The minimum TIMP-1 promoter sequence demonstrated the strongest downregulation in promoter activity following activation of transfected astrocytes, suggesting the location of a silencer element. These data are important for unraveling the mechanisms underlying astrocyte responses during chronic inflammation and have broader implications in other inflammatory diseases that involve MMP/TIMP imbalance. PP02-12 Double-stranded RNA induces the expression of retinoic acidinducible gene-I in cultured astrocytes T. Imaiuzmi, H. Yoshida and K. Satoh Department of Vascular Biology, Hirosaki University School of Medicine, Hirosaki, Japan Chemokine generation in astrocytes may be implicated in immune reactions in the central nervous system. We have studied the effect of double-stranded RNA on the expression of RIG-I in astrocytes. Polyinosinic-polycitydylic acid (poly IC), authentic double-stranded RNA that mimics viral infection, was found to induce the RIG-I expression in an astrocytic cell line, U373-MG. Poly IC also induced the expression of CCL5, a CC type chemokine, and the inhibition of RIG-I expression by RNA interference resulted in reduced CCL5 expression. We conclude that RIG-I may be involved in innate immunity against viral infection in the central nervous system, in part, by regulating the expression of CCL5 in astrocytes. PP03-01 Activation of Peroxisome Proliferator-Activated Receptorgamma (PPARγ) decreases monocyte migration across Blood Brain Barrier (BBB) in a Rho dependent manner HIV-1 caused neurological impairment is associated with migration of virus-infected monocytes across BBB and macrophage accumulation in the brain. While PPARγ (the nuclear ligand-responsive transcription factor) is known as a mediator of pleiotropic anti-inflammatory pathways, its contribution to endothelial barrier function remains largely unknown. We tested effects of PPARγ stimulation on monocyte migration across BBB in vitro models (primary human brain microvascular endothelial cells, HMVEC, plated on porous membranes). Application of tumor necrosis alpha (TNF-α) and monocyte chemoattractive protein-1 (MCP-1), a relevant cytokine and chemokine known to increase during HIV-1 infection, augmented monocyte migration by 2.5-fold across BBB. Pretreatment of endothelial monolayers with rosiglitazone, a PPARγ agonist, diminished by ∼ 5-fold the migration of HIV-1 infected monocytes across BBB compared with untreated controls. Similarly, HMVEC pre-treatment with TNF-α increased adhesion of HIV-1 infected monocytes (3-fold) , and adhesion was reduced by 42% after simultaneous application of rosiglitazone and TNF-α. Since our previous work indicated that monocyte-HMVEC interactions result in activation of Rho, small GTPase, we tested the idea that increased monocyte migration could be mediated by TNF-α induced Rho activation, and PPARγ agonist will prevent these effects. Taken together, these results suggest a novel role for PPARγ agonist in modulation of monocyte-endothelial interactions mediated in part through Rho activation. Utilization of PPARγ agonists approved for clinical use may offer new therapeutic approach in preventing BBB dysfunction seen in HIV-1 brain infection. PP03-02 Inhibition of leukocyte recruitment blocks seizures and epilepsy The pathogenetic link between repeated seizures and epilepsy is completely unknown and this prevents the implementation of successful therapies. The GOAL of this study was to determine a potential role for leukocyte recruitment in the pathogenesis of epilepsy. Intravital microscopy was performed in brain vessels postseizures. Immunofluorescence, immunohistochemistry, electron microscopy, magnetic resonance imaging, telemetry, behavioral evaluation and cognitive evaluation based on enriched open-field revealed were also performed. Results: In humans we observed granulocytes and T cells accumulation into the epileptic brains. We next verified whether leukocyte recruitment plays a role in the pathogenesis of a widely used experimental model of epilepsy. We found increased permeability, vasodilatation and expression of VCAM-1 after status epilepticus (SE) and repeated seizures. Electron microscopy and magnetic resonance imaging show that granulocytes and T cells migrate into the brain post-seizures. Intravital microscopy shows that blockade of alpha4 integrins and VCAM-1 inhibits granulocytes and Th1 lymphocytes rolling and arrest in brain vessels. Therapeutic treatment of mice with anti-alpha4-integrin mAb leads to a drastic reduction of seizures. Moreover, cognitive evaluation revealed that anti-alpha4 treated animals present a significant preservation of the behavior compared to epileptic mice. Strikingly, preventive administration of anti-alpha4integrin therapy, inhibits induction of SE and completely blocks recurrent seizures and development of epilepsy. Conclusion: The results demonstrate an unexpected key role for leukocyte recruitment in the pathogenesis of epilepsy and that anti-leukocyte adhesion therapy has preventive and therapeutic effect in a human model of epilepsy. Background: Although the development of multiple sclerosis (MS) plaques is mainly driven by a Th1 type inflammatory response, the exact mechanism how such restricted populations of T lymphocytes can penetrate into CNS parenchyma is still obscure. Materials and methods: We developed a novel system to evaluate the characters of T lymphocytes attached on and intruded into and beneath human brain microvascular endothelial cell (BMEC) monolayer, using pseudo-3D analysis with confocal laser microscopy. Lymphocytes were obtained from healthy volunteers and MS patients at acute stage. CD4 + lymphocytes were selected and seeded on the TNFα-stimulated human BMEC monolayer. After 6 h, cells were fixed, immonostained, and then the proportion of CD4 + CXCR3 + lymphocytes (representing Th1 cells) and CD4 + CCR4 + lymphocytes (Th2 cells) (1) attached on the surface of BMEC monolayer and (2) migrated into and beneath BMEC monolayer was quantitatively analyzed using this system. Results: In healthy volunteers, lymphocytes migrated beneath BMEC monolayer were significantly Th2 dominant. Conversely, in MS patients at acute stage, most of the Th2 cells stayed on the surface of monolayer and migrated/intruded lymphocytes were significantly Th1 dominant. Conclusion: Intrusion of T cells into CNS parenchyma might be regulated at the blood-brain barrier and this mechanism is possibly impaired in acute MS patients. PP03-04 Astrocytes maintain blood-brain barrier integrity through the production of Angiotensin II Wosik K 1 , Devillers-Dodelet A 1 , Berthelet F 2 , Prat A 1The blood-brain barrier (BBB) restricts the entrance of blood-borne molecules and cells into the central nervous system (CNS), thereby regulating its homeostasis. Astrocytes ensheath brain endothelium and provide crucial factors for the maintenance of the BBB. Multiple sclerosis (MS) is characterized by a breach in BBB integrity. The object of this study was to determine which factors produced by astrocytes contribute to BBB function and integrity. Using human CNS-derived endothelial cells and astrocytes, we have demonstrated that astrocytes express Angiotensinogen (AGT) and produce Angiotensin II, while human brain endothelial cells (HBECs) express angiotensin receptors AT 1 and AT 2 , both in vitro and in situ. The addition of astrocyte conditioned media, Ang II or AT receptor agonist to HBEC cultures induced an important decrease in monolayer permeability as well as specific changes in phosphorylation of the tight junction protein occludin and its concentration into lipid rafts, where tight junctions are assembled. Furthermore, this effect of astrocyte conditioned media could be blocked by the addition of AT 1 but not AT 2 receptor antagonist. The presence of inflammatory cytokines IFNγ and/or TNFα in the astrocyte cultures blocked their ability to produce Ang II. We conclude from these studies that astrocyte-derived angiotensin II, acting via the AT1 receptor, is necessary for BBB integrity. PP03-05 The bioactivity of interferon-beta in Mx-congenic mice Interferon (IFN)-beta is the first and still leading therapeutic intervention shown to change the cause of relapsing remitting multiple sclerosis. However, the mechanism of action of IFN-beta remains in question. The prevailing view is that the benefit from the treatment can be ascribed to IFNbeta's systemic effects. This study investigates the permeability of the blood-brain barrier (BBB) to IFN-beta and thereby, questioning if IFN-beta potentially has an effect within the CNS between relapses.Clinically, the type I IFN-induced MxA gene is used to monitor IFNbeta treatment. We used the mRNA transcript of the murine-counterpart, the Mx1 gene, as a biomarker for IFN-beta treatment of Mx-congenic mice. Using real-time PCR we showed a peak in the level of Mx1 mRNA in white blood cells 3 h after subcutaneous administration of human IFN-beta-1a compared to the denaturated protein and the constitutive level. Analysis of a dose-response relationship and the biodistribution of IFN-beta showed a clear relationship and increased levels of Mx1 mRNA in various organs including; heart, kidney, liver, lung, and spleen.Finally, the permeability of the BBB to IFN-beta was investigated by examining the level of Mx1 mRNA in the perfused CNS. We demonstrated an induction of high levels of Mx1 mRNA, which indicate that IFN-beta crosses the BBB. PP03-06 Diapedesis of monocytes is associated with matrix metalloproteinase (MMP)-mediated occludin disappearance in brain endothelium The blood-brain barrier (BBB) controls the entry of circulating molecules and cells into the brain and is characterized by the presence of endothelial cells connected by tight junctions. Inflammatory cell trafficking into the brain complicates several neurological disorders and conceivably depends on mechanisms that regulate tight junction opening. A detailed study of tight junction dynamics during immune cell diapedesis has been lacking. Therefore, we retrovirally expressed the tight junction protein occludin N-terminally tagged to green fluorescent protein (GFP) in rat brain endothelial cells. Confocal microscopy analyses revealed that GFP-occludin colocalized with the intracellular tight junction protein, ZO-1, and was completely absent at cell borders lacking apposing cells. Mutant cells retained endothelial properties including cell morphology, endothelial markers, proliferation rate and adhesive capacity. By means of live cell imaging studies we show that monocytes scroll over the brain endothelial cell surface towards cell-cell contacts, induce gap formation, which is associated with local disappearance of GFP-occludin, and subsequently traverse the endothelium in a paracellular fashion. Interestingly, occludin contains a putative extracellular MMP cleavage site and using the broad spectrum MMP-inhibitor BB-3103 we show that gap formation, loss of occludin and diapedesis were indeed dependent on the activity of matrix metalloproteinases.Our results provide a novel insight into the mechanism by which leukocytes traverse the BBB and illustrate that therapeutics aimed at the stabilization of the tight junction may be beneficial to resist a neuroinflammatory attack. PP03-07 Highly purified lipoteichoic acid from Gram-positive bacteria induces in vitro blood-brain barrier disruption through glia activation: Role of pro-inflammatory cytokines and nitric oxide The co-culture of bovine capillary endothelial cells and rat primary glial cells was established as an in vitro blood-brain barrier (BBB) model to investigate the mechanisms by which the Gram-positive bacterial cell wall components lipoteichoic acid (LTA) and muramyl dipeptide (MDP) induced injury of BBB structure and function. We found that highly purified LTA disrupted BBB integrity in a concentration-and time-dependent manner indirectly, through glia activation. Low trans-endothelial electrical resistance (TEER) and high permeability to fluorescein isothiocyanate-inulin observed in the presence of LTA-activated glial cells were potentiated by MDP and could be reversed only when glial cells were activated with LTA at 10 μg/ml but not with higher LTA concentrations (30 μg/ml). Immunocytochemistry analysis revealed no evident changes in the distribution of the cytoskeleton protein F-actin and tight junction protein occludin and claudin after LTA treatment. However, the tight junction associated protein AHNAK clearly revealed the morphological alteration of the endothelial cells induced by LTA. LTA-activated glial cells produced nitric oxide (NO) and proinflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) that contributed to LTA-induced BBB disruption, since the direct treatment of the endothelial monolayer with TNF-α and IL-1β increased BBB permeability, whereas the pre-treatment with antibodies against these two cytokines blocked LTA effects. Additionally, NO was also involved in BBB damage, since the NO donor itself (diethylenetriamine-nitric oxide adduct) increased blood-brain barrier permeability and inducible nitric synthase inhibitor (1400 W) partially reversed LTA-induced decrease of trans-endothelial electrical resistance. PP03-08 Altered adrenomedullin and endothelin-1 expression during EAE Barker S a , Mongru R a , Bolton C b , and Paul C c a Queen Mary University of London, b William Harvey Research Institute, London, c University of West England, Bristol; UK Loss of normal blood-brain barrier (BBB) function is an integral feature of the neuroinflammatory process in the human demyelinating disease multiple sclerosis (MS). Adrenomedullin (AM) acts as a vasodilator in many vascular beds including in the cerebral circulation. In vitro work has shown that the peptide can regulate BBB characteristics including transendothelial electrical resistance, permeability and p-glycoprotein pump activation. Furthermore, our preliminary studies demonstrated an elevation of AM during late acute disease in EAE. This study has more fully examined AM expression plus assessed ET-1 changes during EAE.AM and ET-1 levels mRNA were determined in the cerebellum, medulla-pons and spinal cord of Lewis rats inoculated for EAE at height of disease and in early recovery. RT-PCR was carried out on total RNA extracted from rat tissue. AM mRNA remained altered into the recovery phase. The inverse relationship between AM and ET-1 was seen in selected tissues at both timepoints, but was not a feature across all areas, indicating that AM changes may not purely arise from the vascular bed during neuroinflammation.Altered synthesis of AM and ET-1 during neuroinflammatory disease may be of relevance to multiple sclerosis. PP03-09 Development of a physiologic cytokine-activated in vitro blood brain barrier for studying leukocyte diapedesis ex vivo Objective: To develop a physiologic cytokine-activated in vitro blood-brain barrier (aIVBBB) model for studying chemokine-driven leukocyte migration during neuroinflammation. Methods: SV40 T-antigen immortalized human brain microvascular endothelial cells (THBMECs) were cultured to confluence and treated with various concentrations of TNF-α and IFN-γ for 24 h. CCL2 production, a marker of endothelial activation was measured by ELISA, and cell viability ascertained by phase contrast microscopy and Annexin V immunocytochemistry. Transendothelial electrical resistance (TEER) was measured in collagen-coated Transwell™ inserts. Following deduction of physiologic endothelial activation, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 expression were studied by flow cytometry, with fibronectin connecting segment (FN CS)-1 expression deduced by immunocytochemistry. 10 U/mL TNF-α and 20 U/mL IFN-γ (10:20 aTHBMECs) maximally produced CCL2, without compromise to cell viability. There was no change in TEER between the resting and 10:20 aTHBMECs (∼100 Ω cm 2 ), with a progressive decline in resistance with higher cytokine concentrations due to loss of cell-cell contacts secondary to apoptosis-induced cell retraction and death. There was a 450% increase in ICAM-1 and 80% increase in FN CS-1 THBMEC surface expression, with minimal VCAM-1 expression at rest or following physiologic cytokine activation. Conclusions: The 10:20 aTHBMECs form a physiologic aIVBBB that recapitulates the in vivo cerebral microvascular inflammatory milieu. This model can be used to accurately assess chemokine-driven leukocyte transmigration ex vivo as a means of understanding mechanisms and devising potential therapies for neuroinflammation. Purpose: In autoimmune disorders of peripheral nervous system (PNS) such as Guillain-Barré syndrome and chronic inflammatory demyelinating polyradiculoneuropathy, breakdown of blood-nerve barrier (BNB) has been considered as a key step in the disease process. BNB is also an obstacle for drug delivery in PNS disorders. Hence, it is important to know cellular property of peripheral nerve microvascular endothelial cells (PnMECs) constituting bulk of the BNB. Although many in vitro models of bloodbrain barrier (BBB) have been established, very few in vitro BNB models have been reported. So, we established new in vitro BNB models using transgenic rat harboring temperature-sensitive SV40 large T-antigen gene (tsA58 rat) and investigated whether these models posses characteristics as BNB. Method: PnMECs and pericytes were isolated from sciatic nerves of tsA58 rat according to a previously described procedure. PnMECs were identified by the following four criteria: spindle fiber-shaped morphology, immunoreactivity to anti-factor VIII antibody, formation of tight junction and uptake of DiI-Ac-LDL. Pericytes are identified by their peculiar morphology and expression of α-smooth muscle actin. Results: We successfully isolated PnMECs and pericytes from tsA58 rat and these cells are conditionally immortalized nature. PnMECs showed spindle fiber-shaped morphology and expressed uptake of DiI-Ac-LDL, immunoreactivity to anti-factor VIII antibody and barrier properties. Consequently, we built complete in vitro BNB models comprised of PnMECs and pericytes. These models might facilitate analyses for pathophysiology of autoimmune diseases of PNS, moreover, prediction of in vivo drug transportation into PNS parenchyma. PP03-11 Evidence for differential changes of interendothelial junctions in murine neurocysticerosis dependent upon CNS vasculature J.I. Teale 2 1 University of Texas Health Science Center at San Antonio, San Antonio, USA; 2 University of Texas at San Antonio, San Antonio, USAThe delicate balance required to maintain homeostasis of the CNS (central nervous system) is controlled by the BBB (blood brain barrier) which impedes influx of most compounds from the blood to the CNS. However, our group has recently shown in a murine model of neurocysticerosis (NCC) that BBB disruption varies depending upon the anatomical site/vascular bed being analyzed in the CNS.In this study further understanding of the mechanisms of BBB disruption were explored in blood vessels located in leptomeninges (pial vessels) and brain parenchyma (parenchymal vessels) by examining interendothelial junction proteins involved in the integrity of the BBB. Mice were infected intracranially with the parasite Mesocestoides corti and sacrificed at various times post infection. Different anatomical areas of infected brain were analyzed by three color immunofluoresence utilizing antibodies against tight junction (TJ) and adherens junction (AJ) proteins. The results showed increased permeability in vessels with structural alterations, disappearance, or apparent proteolysis of particular TJ and AJ proteins. The extent and timing of these changes differed between pial vessels of the meninges and vessels of the parenchyma (pial vessel disruption within days versus weeks for parenchymal vessels).To approach potential mechanisms, the expression and activity of individual matrix metalloproteinases (MMP) was evaluated by in situ zymography at different times post infection. Moreover, the timing of MMP activity in pial and parenchymal vessels correlated with the different kinetic breakdown of these vessels. PP03-12 Characterising autoreactive T lymphocyte migration and infiltrationa bird's eye view K.R. Forrester 2 and C. Linington 1 Department of Medicine and Therapeutics 1 and Ophthalmology 2 , Institute of Medical Sciences, University of Aberdeen, Aberdeen AB252ZD, Scotland UK Studies using central nervous system (CNS) antigen specific T cell lines engineered to express GFP have dissected the migratory pathways and activation of lymphocytes during adoptive transfer EAE, an animal model of MS. However these studies are hindered by the difficulty of identifying the initial sites of infiltration in such a complex target organ. To overcome this problem we have utilised another model of autoimmunity, Experimental Autoimmune Uveoretinitis (EAU), in which the target organ is smaller and the location of lymphocyte infiltration is easier to dissect. We generated retinal antigen specific GFP expressing T lymphocytes and tracked their migration to the eye in Lewis rats. We recovered cells from various lymphoid organs and examined their activation status. We characterised the infiltration of these cells into the eye, defining the precise locations of entry and studying macrophage infiltration and antigen presentation. Surgical manipulation and injection of soluble antigen enabled us to dissect the mechanisms further. Our results demonstrate that following transfer of activated GFP T lymphocytes organ specific accumulation was not observed until two days after transfer when the cells reappeared in the spleen in large numbers in a non-activated state. On day three the cells migrated via the blood (still non-activated) and started appearing in the eye where they strongly express activation markers. This is in concurrence with published observations in EAE and demonstrates that these mechanisms are not specific to particular diseases. PP04-01 The homeostatic chemokine CCL19 is constitutively expressed in the CNS, up-regulated in multiple sclerosis lesions, and is correlated with intrathecal IgG production This study focused on the homeostatic chemokines CCL19 and CCL21, which both bind to CCR7 and direct B cells, mature dendritic cells, and a subset of T cells to secondary lymphoid organs. We report that in the normal human brain CCL19 is transcribed, detectable in tissue lysates, and displayed on endothelial cells. In both active and inactive MS lesions CCL19 transcripts were elevated and strong CCL19 immunostaining of astrocytes and blood vessel endothelial cells was seen. In cerebrospinal fluid (CSF) from relapsing remitting and secondary progressive MS patients CCL19 was elevated and correlated with intrathecal IgG production. CCL21, the other CCR7 ligand, was not detected in control or MS CSF samples, and quantitative PCR and immunostaining showed only very low levels of this chemokine in MS lesions. Together, this study suggests a role of CCL19 in immunosurveillance of the CNS and maintenance of immune cells in MS brains. Active multiple sclerosis (MS) is characterized by the presence of perivascular inflammatory foci localized in the central nervous system (CNS). Inflammatory cells forming those foci migrate from the blood to the CNS. Several studies confirmed that chemokines and their receptors play an important role in that process. The major goal of this study was to analyze the migratory activity of subpopulations of peripheral blood mononuclear cells (PBMC) from the blood of MS patients stimulated by chemokine CCL5/RANTES. Moreover the impact of MS treatment with methylprednisolone (active MS) and mitoxantrone (progressing MS) on CCL5-induced chemotactic activity of PBMC subpopulations was analyzed. Chemotactic activity of mononuclear leukocytes was measured in vitro in Neuroprobe MBA96 chemotaxis chamber using fluorimetric reader. We observed that in active MS before any treatment in vitro chemotactic activity of lymphocytes after stimulation with CCL5 was significantly increased. Spontaneous migration of lymphocytes was similar in all studied groups. Treatment of MS with methylprednisolone and mitoxantrone diminished this activity to the level observed in control groups of patients with other neurological diseases (OND) and healthy controls (HC). We did not observe any significant changes in spontaneous and stimulated by CCL5 migratory activity of monocytes from MS patients. Our results suggest that in active MS chemokine-induced migratory activity of lymphocytes is increased. Objective: Acute cerebral ischaemia induces local inflammatory reaction including expression of chemokines, which precedes relevant leukocyte infiltration contributing to tissue injury. The objective of the study was to test hypothesis that CXCL1 and CXCL6 chemokines, potent neutrophil chemoattractants, play a role in inflammation during early phase of ischaemic stroke. Methods: The CXCL1 and CXCL6 levels in the CSF and serum obtained during 24 h from 23 ischaemic stroke patients aged 72.2 +/− 10.8 years have been measured by ELISA. CSF and blood samples from 15 tension headache patients served as a control group. The neurological stroke severity was estimated with Scandinavian Stroke Scale (SSS) within 24 h of stroke (SSS-1) and two weeks later (SSS-2). Results: CXCL1 and CXCL6 levels were significantly elevated in the CSF of patients with ischaemic stroke in comparison with controls (65.6 +/ − 22.3 pg/ml vs. 43.8 +/− 2.3 pg/ml for CXCL1 and 3.1 +/− 0.9 pg/ml vs. 1.8 +/− 0.7 pg/ml for CXCL6). Serum levels of studied chemokines did not differ from control values. CSF CXCL1 and CXCL6 levels correlated significantly with the neurological stroke severity within 24 h and after two weeks from the onset of stroke. This is the first such observation. Conclusions: Our results suggest that CXCL1 and CXCL6 chemokines may play a role in the inflammatory reaction during early phase of ischaemic stroke and CSF CXCL1 and CXCL6 levels are associated with stroke severity and have predictive value for short term stroke outcome. It is well documented that chemokines play an important role in development of inflammatory foci through regulation of cell trafficking within tissues. This multistep process occurs also within the central nervous system (CNS) in many pathological conditions. In a present study we have investigated the role of chemokines and their receptors in the development of CNS inflammation using previously described model of local CNS inflammation. In this model inflammatory focus develops after deposition of BCG (Bacillus Calmette-Guerin) into the brain parenchyma followed by subcutaneous peripheral injection of BCG four weeks later. Accumulation of inflammatory cells around the site of BCG deposition is followed by a subsequent myelin damage. Expression of inflammatory (CCL5) and constitutive (CCL19, CCL21, CX3CL1) chemokines as well as chemokine receptors CCR5, CCR7 and CX3CR1 was analyzed. We performed RNA expression analysis using quantitative RPA assay. Pathological features of inflammation were confirmed by analysis of expression of many specific markers of inflammatory cell subpopulations. Within 7 days post peripheral challenge we have observed an increase of CCL19 expression in a hemisphere injected with BCG. Interestingly in 10 days post peripheral challenge subsequent increase in contralateral hemisphere was also observed. A similar pattern of expression was also observed for chemokine CCL5. In contralateral hemisphere increase of this expression was observed 2 weeks after peripheral challenge. Our results confirm that some chemokines and their receptors play an important role in the control of cell trafficking during CNS inflammation. PP04-05 Astrocyte-specific CXCL1 overproduction may be neuroprotective during CNS demyelination K. M. Omari a , and C. S. Raine a a Department of Pathology, Albert Einstein College of Medicine, Bronx, USA Factors responsible for oligodendrocyte survival, a feature observed around early and active multiple sclerosis (MS) lesions, are not fully elucidated. Existing evidence in rodents demonstrate that the chemokine, CXCL1, induces proliferation and inhibits chemotaxis of oligodendrocyte precursor cells (OPCs). To investigate the effects of CXC chemokines during the course of central nervous system (CNS) inflammatory responses, we generated CXCL1/GFAP double-transgenic (Tg) mice, which overexpress CXCL1 inducibly under the control of the astrocyte specific gene, glial fibrillary acidic protein (GFAP). Double (CXCL1/ GFAP)-Tg, single (CXCL1)-Tg and wildtype (wt) animals were sensitized with myelin oligodendrocyte glycoprotein (MOG) for experimental autoimmune encephalomyelitis (EAE). After onset of clinical signs, CXCL1 production was initiated by intraperitoneal doxycycline injection (500 μg/animal). Preliminary results show that double-Tg animals display much milder clinical disease. Histopathology revealed that although inflammation was present in spinal cord and optic nerve tissue of double-Tg, single-Tg, and wt control animals by 15 days post-immunization (d.p. ), demyelination and axonal pathology (Wallerian degeneration, WD) were more prominent in the control mice. By 40 d.p.i., inflammation and demyelination were significantly diminished in double-Tg mice, compared to controls, and WD was markedly less. Interestingly, relative to the levels of myelin and axonal pathology, remyelination was greater in the double-Tg group, and there was an associated increase in oligodendrocytes, suggestive of an oligodendroglial response. Ongoing protein expression studies are examining inflammatory infiltrates, OPCs, and proliferation (BrdU incorporation) within the CNS. Thus, preliminary findings suggest a role for CXCL1 in exerting a neuroprotective role during the course of EAE.Supported in part by NMSS RG 1001-K-1, NS 08952, NS 11920, NS 07098 and the Multiple Sclerosis Foundation. PP04-07 Transcriptional regulation of CCR5 in astrocytes and microglia Several chemokines and chemokine receptors, including CCR5, are implicated in the pathogenesis of multiple sclerosis (MS). Earlier, we have shown that MS lesions are hallmarked by enhanced expression of "stress-response" transcription factors such as IRF-1, NF-κB and CREB, leading to enhanced expression of both classes of MHC molecules. The expression of these molecules overlaps with the expression of CCR5 in MS lesions. Therefore, we investigated whether these factors are also involved in the transcriptional regulation of CCR5.Using promoter evaluation assays such as electrophoretic mobility shift assay and transient transfection, we determined that neither IRF-1 nor NF-κB is involved in the activation of the CCR5 promoter. Additionally, we show that these factors are not involved in the induction of transcription of endogenous CCR5 in human primary microglia and astrocytes, as determined by RT-PCR. In addition, our data also suggest that epigenetic mechanisms might be implicated in the transcriptional control of CCR5.Taken together, we conclude that expression of CCR5 is regulated by several general, but distinct regulatory mechanisms and that alterations in these mechanisms could account for the aberrant expression of CCR5 in MS lesions.Supported by the Dutch MS Research Foundation (grant 00-407 MS/04-543 MS). PP04-08 Differential chemokine expression after neuronal injury Chemokines are discussed to be involved in the signalling between damaged neurons and microglia. We have recently shown that the chemokine CCL21 is upregulated in neurons after cellular injury in vitro and in vivo (Biber et al., 2001; de Jong et al., 2005) and attracts microglia via the chemokine receptor CXCR3 (Rappert et al., 2002) . In CXCR3 knockout animals microglial activation after injury was almost absent and survival of secondary neurons was much higher when compared to controls (Rappert et al., 2004) .Neurons also produce the chemokine CXCL10 which is also a ligand for microglial CXCR3, raising the question why neurons would express two ligands for one receptor. Accordingly, we have investigated and compared the expression and distribution pattern of CXCL10 with that of CCL21 in cultured neurons after glutamate treatment.Both chemokines are packed in vesicles. However, whereas CCL21 protein is induced only after neuronal damage, CXCL10 is expressed constitutively at high levels and is not regulated by neuronal damage. Furthermore, CCL21 is released from damaged neurons, whereas we did not yet find conditions that would induce the release of CXCL10 in damaged neurons. These results show for the first time that two neuronal chemokines that act on the same receptor in microglia are differentially regulated by neurons. Whether both CCL21 and CXCL10 are involved in neuron microglia signaling and whether both chemokines might induce different effects in microglia remains to be established. Biber K, et al., (2001) Objective: A glial reaction associated with the up-regulation of inducible nitric oxide synthase (iNOS) has been suggested to play an important role in an inflammatory process of neurodegenerative diseases. However, the mechanism how iNOS is induced in glial cells is not clearly understood. To clarify the mechanism of the induction, we focused on class IV semaphorin Sema4D/CD100 which is known to enhance CD40 signaling in B cells and dendritic cells in the immune system.Methods: The expression of Sema4D and its putative receptors, CD72 and Plexin-B1, on primary neural cells was investigated by RT-PCR and Western blot analysis. The effect of Sema4D on the induction of iNOS was investigated by incubating primary astrocytes and microglia with recombinant Sema4D.Results: Sema4D and its receptors were expressed in primary neurons, astrocytes and microglia. This enhancement was also observed in glial cells prepared from mice deficient for CD72. Sema4D enhanced the ERK1/2 activation by CD40 stimulation and an inhibition of ERK1/2 pathway with U0126 partially decreased iNOS expression in microglia.Conclusion: Sema4D/CD100 and its receptors CD72 and Plexin-B1 are expressed on neural cells and could exacerbate neuroinflammatory process by enhancing iNOS expression. Plexin-B1 at least in part participates in this process via ERK1/2 pathway. PP05-02 Temporal genetic pathways in thymic selection reveal shared regulatory networks with neuronal degeneration and apoptosis Given their shared requirements for the generation of complexity, flexibility, plasticity, memory and specificity, the brain and immune systems might have more in common than first meets the eye. We have recently demonstrated that L-glutamate, a major excitatory amino acid in synaptic transmission in the nervous system, acts as an immunotransmitter at the immunological synapse formed between T and dendritic cells, contributing to calcium mobilization and T cell apoptosis during thymic negative selection. To further explore the compared physiology between the immune and nervous system, we combined genome-wide expression analysis with a new bioinformatics method to identify functional networks involving Nur77 in thymic negative selection. We used Ingenuity Pathway Analysis, a new web-based entry tool, which provides molecular networks with a high degree of connectivity over all genes documented in the literature. A systematic temporal expression profiling of thymocytes from TCR transgenic mice undergoing thymic negative selection was generated using Agilent oligonucleotide microarrays. The level of expression of a 44,000 probe set representing around 22,000 mouse known genes was monitored with 9 kinetics time points over a 24 h period, with a focus on 6 early time points over 3 h. Dynamically regulated genes identified using Resolver software were overlaid onto the cellular pathway maps in the Ingenuity Pathway Analysis software application to generate very significant high-scoring networks, including Nur77 in the early time points. In addition to documenting genetic pathways involving Nur77 in thymic negative selection, our findings provide new insights into the regulation of thymocyte apoptosis as it relates to neuronal apoptosis and neurodegeneration. PP05-03 Astrocytes as regulators of human CNS inflammation Alex Kostianovsky and David E. Anderson Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, USA A growing body of literature indicates that resident glial cell populations can modulate immune responses within the central nervous system (CNS). When monocytes are co-cultured with primary astrocytes in the presence of LPS, monocyte secretion of TNF-α is completely inhibited, whereas there is no inhibition when CD40 ligand is the stimulus. These data suggest that within the human CNS, pathogenmediated inflammation (LPS) is, at least in part, naturally inhibited by astrocytes, whereas T cell-mediated inflammation (CD40 ligand) is not. This observation has relevance to regulation of T cell-mediated inflammation within the CNS, including that associated with multiple sclerosis and responses to brain tumors. Indeed, using a transformed primary malignant glioma (Glioblastoma Multiformae or GBM) cell line, we have found that, in contrast to non-transformed primary human astrocytes, GBM tumor cells acquire the ability to suppress monocyte secretion of TNF-α in response to both LPS and CD40 ligand stimulation. Transwell assays indicate that both soluble and cell-contact dependent mechanisms are responsible for tumor cell-mediated suppression of monocyte activation, and neutralization studies exclude TGF-β, IL-10, COX 2 and CD200 as molecules associated with this suppression. A highthroughput screening strategy is planned to identify the relevant pathway (s).Collectively, these results indicate that astrocytes have an inherent regulatory capacity within the CNS that is responsive to nature of the inflammatory stimulus. PP05-04 Neuronal antigen-containing antigen-presenting cells in cervical lymph nodes of multiple sclerosis patients and EAE animals are functionally distinct from myelin antigen-containing cells Multiple sclerosis (MS) is characterized by demyelination and neuronal damage within the central nervous system (CNS). Transfer of CNS antigens to the draining cervical lymph nodes (CLN) likely is crucial in initiating or modulating immune responses against myelin, but also against neurons. Although myelin antigens have been demonstrated in CLN of MS patients, the presence of neuronal antigens in CLN of MS patients has not yet been determined. We here report the presence of neuronal antigens in CLN from MS patients, common marmoset monkeys, rhesus monkeys, and Biozzi ABH mice with EAE. Draining lymph nodes (lumbar lymph nodes and deep CLN) of cuprizone-treated mice, exhibiting extensive CNS demyelination with little neuronal damage, contained abundant myelin antigens but not the neuronal antigen neurofilament light. In human CLN, cells containing the neuronal antigen MAP-2 expressed the APC markers MHC class II and CD40, and the proinflammatory molecules IL-12p40/p70 and TNF-α. In contrast, the majority of cells containing the myelin antigen MOG expressed the anti-inflammatory molecules IL-1 receptor antagonist and TGF-â. 20% of MOG-containing cells expressed the lymph node homing molecule CCR7, whereas CCR7 expression was absent from MAP-2-containing cells.Using an in vitro model, we confirmed increased CCR7 mRNA expression by human monocyte-derived macrophages following human myelin ingestion in vitro. We hypothesize that the specific immunophenotype of the APC may be caused by the nature of the phagocytozed antigen or the micro-environment at the location of antigen uptake. PP05-05 Cross-presentation of central nervous system-derived antigen induces robust priming of CD8 + T cells in cervical lymph nodes Lisa Walter and Matthew L. Albert Institut Pasteur, Paris, France The efficiency of antigen presentation from the central nervous system (CNS) to initiate and induce antigen-specific cytotoxic CD8 + T cell immune responses remains unclear. To determine how presentation of antigen from the brain versus from other tissues differs in inducing a cytotoxic CD8 + T cell response, antigen was administered intracerebrally (IC), subcutaneously (SC), or intravenously (IV) into mice. Mouse splenocytes deficient in major histocompatibility complex class I H-2K b and expressing the membrane-bound ovalbumin antigen (mOVA/H-2K b−/ − ), were injected to monitor responses to cross-presentation of the H-2K b restricted epitope, SIINFEKL. IC-introduced mOVA/H-2K b−/− splenocytes induce higher in vivo CD8 + T cell killing, interferon gamma production, and proliferation than when administered SC or IV. CD8 + T cell priming from cross-presented IC antigen occurs in the cervical lymph nodes and requires CD4 + T cell help. In addition, CD8 + T cell priming due to crosspresentation, but not direct presentation, requires CD40 expression by the host. Taken together, these data imply that an immune response resulting from cross-presentation of IC antigen, once initiated, is more efficient and therefore more likely to induce immune priming compared with responses initiated elsewhere in the body. Similar to other tissues, antigen crosspresentation from the CNS requires CD40 expression by the host, presumably mediating helper-dependent cross-priming of CD8 + T cells specific to CNS-derived antigen. PP05-06 Antigen specific activation of naïve CD8 T cells by resident antigen presenting cells in the brain: The role of brain antigen presenting cells in the initiation of CNS inflammation The compartments in which critical accessory cell/T cell interactions take place prior to the onset of neuroinflammation have not yet been clarified. In this study we show that naïve T cells can be activated by local resident accessory cells in nervous tissue in the absence of peripheral immune organs. To define CNS-restricted naïve T cell activation, we prepared brain slice cultures from experimental animals and maintained these tissues in artificial cerebral spinal fluid for several days. One hour prior to the preparation of brain slice cultures, mice were injected intracerebrally with ovalbumin antigen. When naïve OT-1 ovalbumin-specific CD8 + T cells were added to these cultures, these T cells become activated, proliferated and migrated into the brain slices which contained cognate antigens. Brain resident dendritic cell-like accessory cells (CD11c + ) were also mobile and able to migrate from the brain slices in the presence of inflammatory chemokine, MIP-3β. Proliferation of OT1 antigen-specific T cells and the migration of brain resident accessory cells were abrogated by the deletion of CD11c + cells from Itgax-DTR/EGFP mice prior to the generation of brain slices. Due to the transgene expression, CD11c expressing cells can be selectively depleted (huDTR) and/or identified (GFP fluorescence) in these mice. PP05-08 Metalloproteinases control brain inflammation induced by pertussis toxin in mice overexpressing the chemokine CCL2 in the CNS Leukocytes accumulate spontaneously in the perivascular space in brains of transgenic (Tg) mice that overexpress CC chemokine ligand 2 (CCL2) under control of a CNS-specific promoter. Pertussis toxin (PTx) given intraperitoneally induced encephalopathy and weight loss in Tg mice. We used flow cytometry, USPIOenhanced magnetic resonance imaging and immunofluorescent staining to show that encephalopathy involved leukocyte migration across the glia limitans into the brain parenchyma, identifying this as the critical step in inducing clinical symptoms. Unmanipulated Tg mice had elevated expression of TIMP-1, MMP-10 and 12 mRNA in the brain. PTx further induced expression of TIMP-1, ADAM-12 and MMPs 8 and 10 in brains of Tg mice. Levels of the microglial-associated MP, MMP-15 were not affected in control or PTx-treated Tg mice. PTx also upregulated expression of proinflammatory cytokines IL-1β and TNFα mRNA in Tg CNS. Weight loss and parenchymal infiltration induced by PTx were significantly inhibited by the broad-spectrum MP inhibitor BB-94/Batimastat. Our finding that MPs mediate PTx-induced parenchymal infiltration to the chemokine-overexpressing CNS has relevance for the pathogenesis of human diseases involving CNS inflammation, such as multiple sclerosis. PP05-09 Induction of tolerance for alpha B-crystallin in alpha Bcrystallin knock-out mice Previous data indicate that in humans a strong pro-inflammatory memory T-cell response exists against the CNS myelin antigen alpha Bcrystallin, which qualify it as a potential autoimmune target in multiple sclerosis. In our study we were able to specifically tolerize these proinflammatory memory T-cells for alpha B-crystallin in an animal model. Further efforts are made towards a clinical study.Since in contrast to humans, rodents are naturally tolerant for self alpha B-crystallin, the tolerization studies were performed in alpha Bcrystallin knock-out mice that are highly responsive to the protein antigen and that lack all form of naturally tolerance for alpha B-crystallin. Tolerance-inducing protocols were started 3 weeks after immunization with alpha B-crystallin in CFA. Tolerance was induced by administering intravenous alpha B-crystallin in the tail vein in various dose-timing regiments.Low dose induction of tolerance led to an almost complete abrogation of T-cell responsiveness in lymph node-derived T cells upon subsequent antigenic challenge. Although a limited induction of the T cell response was detected after in vitro re-challenge this was swiftly followed by a state of non-responsiveness. Even a boost with antigen in adjuvant after tolerance induction did not reverse the tolerant state. In contrast to the T-cell response, an increased antibody response was detected. Future efforts will be made to study the tolerance inducing mechanisms such as anergy or regulatory T-cells and the role of antibodies. PP05-10 Immature human dendritic cells express latency-associated peptide (LAP) and inhibit Th1 polarization in a TGF-β dependent fashion Multiple sclerosis (MS) is a chronic Th1 cell mediated inflammatory disease of the CNS. We investigated the role of dendritic cells (DCs) in MS as cells important for initiating an immune response and maintaining peripheral tolerance and found DC activation in the blood of MS patients (J Immunology, in press). The exact mechanism by which DCs are instructing/influencing the generation of regulatory versus effector T cells is not clear. TGF-β is an important immunoregulatory molecule and is implicated in the induction of regulatory T cells. Such neutralization resulted in enhanced T cell proliferation and IFN-γ secretion by allogeneic CD4 + T cells incubated with ex-vivo isolated human DCs. Furthermore, when DCs were matured by stimulation with LPS so they were no longer tolerogenic, we observed the loss of membrane bound LAP, accompanied by up-modulation of HLAII and co-stimulatory molecules CD80, CD83, CD86 and CD40. Furthermore, initial results demonstrate altered tolerogenic properties of DC subpopulations in MS. Taken together, our results indicate that expression of TGF-β on DCs in the form of LAP may play an important role in the tolerogenic properties of immature DCs, which serve to limit or prevent autoimmune responses and this may be abnormal in MS. PP05-11 CD4 + CD25 + Foxp3 + T cells and CD4 + CD25 − Foxp3 + T cells in aged mice T. Nishioka, R. Iida and J. Shimizu Section of Immunology, Department of Mechanism of Aging, National Institute for Longevity Sciences, Obu, JapanAdvancing age is associated with significant alterations in immune functions, including a progressive decline in CD4 T function, in both mice and humans. However, it has been unknown whether regulatory/suppressive CD4 T cells are involved in this decline. Our in vitro analyses revealed that CD4 + CD25 + T cells, the well-characterized naturally occurring regulatory/ suppressive CD4 T cells, in aged mice are functionally comparable to those in young mice (i.e., anergic and suppressive), though slightly increased in number. In contrast, functional changes to whole CD4 + CD25 − T cells were pronounced in aged mice. We separated CD4 T cells into small subpopulations based on the staining with Rhodamine-123 (R123). Cell populations rapidly extruding R123 (referred to as R123 lo cells), the majority of aged CD4 + CD25 − T cells, exhibited a significant hyporesponsiveness, and the remaining cells (CD4 + CD25 − R123 hi T cells) maintained a normal responsiveness. Furthermore, we identified Foxp3 (a transcription factor critical in conferring the regulatory/suppressive function to CD4 T cells)-positive suppressive CD4 T cells among aged hyporesponsive CD4 + CD25 − R123 lo T cells. These results suggest that the age-related decline in T cell-mediated immune responses is ascribable to changes in the CD4 + CD25 − T cell population, and not to a functional augmentation of suppressive CD4 + CD25 + T cells. PP05-12 Lack of VLA-4 expression defines functional immunesuppressive Treg cells in humans Migration and homing is largely controlled by adhesion molecules. We therefore compared expression of various integrins on CD25high regulatory T cells (Treg) and on CD25 low effector CD4 + T cells. On human PBMC a striking difference was observed for CD49d, the ?-chain of VLA-4 (α4β1). CD49d was highly expressed on the CD25 low effector T cells but was down-regulated on most CD25 high cells. The differential expression pattern was evident especially on the CD45RO + CCR6 + effector memory-like T cell subsets, where the loss of VLA-4 was compensated on Treg cells by the expression of CLA (cutaneous lymphocyte antigen). CD45RO + CCR6 + Treg cells therefore bind efficiently to the CLA-ligand E-selectin, whereas CD4 + effector T cells preferentially adhere to the VLA-4 ligand VCAM-1. At least in principle, invasion of inflamed tissues can therefore be regulated in a subset-specific way by selective expression of these ligands at the endothelial barrier.Notably, on CCR6 + CD25 high cells the presence of CD49d correlates with an apparent loss in activity. Foxp3 expressing CD49d high CD25 high cells, present in PBMC of healthy donors as a minor subset, showed only marginal suppression whereas CD49d-CD25 high cells efficiently inhibited the proliferation of CD4 + CD25 − T cells. Thus, the differential expression of VLA-4 and CLA not only allows to selectively recruit effector-memory like Treg (TREM) and conventional effector memory cells (TEM), the absence of CD49d can also be used as key-marker to distinguish functional immune-suppressive Treg cells from other less active Foxp3 + T cells. PP05-13 Control of IL-5 production in CD1d-reactive human CD4 + NKT cell clones from MS patients following exogenous IL-2 co-stimulation K. Sakuishi ab , S. Miyake a , T. Yamamura a a Department of Immunology, National Institute of Neuroscience, NCNP, Tokyo, Japan; b Department of Neurology, University of Tokyo, Tokyo, JapanCD1d-restricted NKT cells can produce both Th1 and Th2 cytokines. Recently, NKT cells were found to have the potential to suppress the development of experimental allergic encephalomyelitis (EAE), opening up the possibility as a therapeutic target for multiple sclerosis (MS). However, how they naturally regulate immune responses in humans remains largely obscure. We have found that, although human CD4 + NKT cell clones cocultured with CD1d + antigen presenting cells (APCs) do not produce cytokines, by adding exogenous IL-2, a significant number of clones selectively and remarkably produced IL-5. Methods: Human CD4 + CD1d-restricted NKT cell clones were established by stimulating fresh PBMC with α-galactosylceramide or OCH, its synthetic analogue, and then sorting for invariant TCR + /Vβ11 + /CD4 + /CD8 − cells. The NKT cell clones were co-cultured with APCs with or without recombinant IL-2, and cytokines in the supernatant were evaluated. Results: When co-cultured with APCs, 8 out of 24 NKT cell clones (including those of MS patients) produced remarkable amounts of IL-5 but little IFNγ in the presence, but not in the absence, of IL-2. CD1d-expressing Hela cells as well as immature DCs served as good APCs, whereas mocktransfected Hela cells did not. CD4 + NKT cells freshly isolated from BALB/ c mice similarly showed a Th2 polarization pattern, supporting the physiological implications of our findings. Conclusion: Taken together, IL-2 plays a key role in triggering the Th2 polarizing potential of CD4 + NKT cells in response to weak endogenous antigen stimulation. We propose a novel IL-2 dependent NKT cell function in immune regulation. PP05-14 Natural killer T cell ligand OCH as a potential therapeutics for multiple sclerosis: Mechanism for OCH-induced Th2 polarization in vivo Shinji Oki, Takashi Yamamura and Sachiko Miyake Department of Immunology, National Institute of Neuroscience, NCNP, Tokyo, Japan Natural killer T (NKT) cells are one of the most potent immune modulators after stimulation with glycolipid ligand such as αgalactosylceramide (αGC) through a massive production of immunoregulatory cytokines including interleukin-4 (IL-4) and interferon-γ (IFN-γ). We have demonstrated that OCH, a sphingosine-truncated analogue of αGC, prevents a variety of experimental autoimmune disease models including experimental autoimmune encephalomyelitis (EAE) by inducing the preferential NKT cell-dependent IL-4 production and subsequent Th2 polarization in vivo. Biochemical and molecular biological analysis revealed that the specific feature of the glycolipid is due to its unstable capture on CD1d and a short-lived stimulation to NKT cells, which is insufficient for effective c-Rel induction followed by the lack of effective IFN-γ production. Intriguingly, OCH induces less IFN-γ production not only by NKT cells but also by natural killer (NK) cells, another major source of IFN-γ after glycolipid administration in vivo. Due to an insufficient primary IFN-γ production and CD40L expression by activated NKT cells, OCH exert marginal IL-12 production by dendritic cells, resulted in lower secondary IFN-γ induction by NKT and NK cells. Accordingly, the availability of IL-12 in connection with microbial infection is one of the critical determinants for the cytokine profile after in vivo administration of OCH. Taken together, OCH has a unique property for selective IL-4 production by NKT cells without inducing IFN-γ production by NKT and NK cells. A proper application of OCH in vivo is beneficial for a NKT cell-targeted therapeutic intervention of Th1-mediated autoimmune diseases such as multiple sclerosis (MS). Recent studies have demonstrated that myelin-reactive IL-17-producing T cells would efficiently mediate the destructive autoimmune pathology of experimental autoimmune encephalomyelitis (EAE). Therefore, the IL-23-IL-17 pathway could be pivotal in the pathogenesis of multiple sclerosis. Although much needs to be learned about the characteristics of IL-17 producing T cells, CCL2 is known to augment the T cell production of IL-17. This allowed us to address if IL-17 producing T cells might express CCR2, the CCL2 receptor. Of interest, CCR2 is highly expressed within MS plaques and CCR2 knockout mice are resistant to EAE. When whole T cells, CCR2 + or CCR2 − T cells purified from healthy donor blood were activated by PMA and ionomycin for 1 day, only CCR2 + T cells produced a large amount of IL-17. In sharp contrast, CCR5 + T cells, but not CCR5-T cells, produced IFN-γ, whereas both of the populations produced IL-17 equally. Notably, most CCR2 + T cells coexpress CCR5, and only a minor population expresses CCR2 alone. We found that the minor CCR2 + CCR5 − fraction was the main source of IL-17. The CCR2 single positive cells also produced IFN-γ, but it was much less than that from CCR2 + CCR5 + fraction. To conclude, we have identified CCR2 + CCR5 − T cells in the human blood as IL-17-producing T cells, which is consistent with that IL-17-producing T cells would develop via a lineage distinct from the Th1 lineage. PP05-16 The role of CD4 + CD28 null T cells in autoimmune diseases The loss of CD28 expression on both CD4 + and CD8 + T cells has been described as a good biological indicator of aging of the immune system. Increased percentages of CD4 + CD28 null T cells have also been associated with a number of pathological conditions. We previously demonstrated elevated percentages of these cells in blood of a subset of healthy controls (HC) and patients with an autoimmune disease. However, the fraction of people with an increased percentage of CD4 + CD28 null T cells was higher in diseased individuals (48/160) as compared to HC (7/50). To unravel the role of CD4 + CD28 null T cells in autoimmune diseases some functional characteristics of these cells have been studied.Intracellular FACS staining demonstrated that a large proportion of the CD4 + CD28 null T cell subset produced IFNγ upon stimulation. The majority of these cells also contained intracellular deposits of cytotoxic molecules. In addition, CD4 + CD28 null T cells could be detected in cerebrospinal fluid of MS patients and synovial tissue of RA patients. To study antigen reactivity of CD4 + CD28 null T cells, an in vitro assay was developed. Peripheral blood mononuclear cells are cultured with an array of allo-and autoantigens like myelin basic protein and human collagen type II. Proliferation and cytokine production will demonstrate antigen reactivity. This assay is currently pending.CD4 + CD28 null T cells represent a cell subset with aberrant functional capacities which can be detected at the site of tissue damage in autoimmune diseases. Unraveling the antigen specificity of CD4 + CD28 null T cells will provide more insight into their role in the pathogenesis of autoimmune diseases. PP05-17 Endoplasmic reticulum-resident heat shock protein gp96 as an innate sensor of damage induced by tissue remodelling, stress and bacterial peptidoglycan Tissue disintegration after injury leads in the endoplasmic reticulum (ER) to activation of adaptive pathways known as the ER stress response. It is directed to correction of unfolded proteins, to the activation of proteasome-dependent ER-associated degradation of the misfolded proteins or to activation of protein translation to modulate the polypeptide traffic into the ER. Since, in these events a crucial role plays gp96, which acts not only as a molecular chaperone but also as an adjuvant, able to induce the specific immune responses against tumours and some bacteria, in this study we analysed its role in conditions of: (1) normal growth, induced by partial hepatectomy, (2) in psychosocial stress without the tissue lesions and (3) after the treatment with bacterial peptidoglycan-monomer (PGM) and PGM-Zn. Tissue expression of Gp96 protein and mRNA was estimated in liver, thymus and spleen and the data were correlated with phenotype and cytotoxicity of hepatic and splenic mononuclear lymphatic cells (MNLC) against the syngeneic thymocytes, NK and LAK-sensitive targets.All procedures induced fast cytoplasmic overexpression of gp96 staining in hepatocytes, followed by surface expression of gp96 on MNLC and upregulation of gp96 mRNA in the spleen and in the thymus. Simultaneously, in the liver accumulated CD3 intermediate /NK1.1 + /CD69 + cells, while hepatic and splenic MNLC became highly cytotoxic against syngeneic thymocytes and YAC-1 and P815, implying that during the disturbance of morphostasis gp96 may serve as a natural adjuvant for chaperoning antigenic self peptides into the immune surveillance pathways, resulting in activation of autoreactive NKT clones with morphogenetic potential (supported by grants from Croatian Ministry of Science). PP05-18 In vivo modulation of cellular and humoral immune response to tetanus toxoid by mesenchymal stem cells S Chiesa 1 , E Traggiai 2 and A Uccelli 1 1 Department of Neuroscience Ophthalmology and Genetic, Genoa, Italy; 2 Institute G.Gaslini, Genoa, ItalyMurine mesenchymal stem cells (MSCs) ameliorate experimental autoimmune encephalomyelitis (EAE), through the induction of peripheral T cell tolerance. Nevertheless, the effect of MSC on the physiological immune response to non self antigens such as tetanus toxoid (TT) is still largely unknown. Thus, we sought analysing the dynamic changes of the humoral and cellular response to TT upon MSCs administration. 1 × 10 6 MSCs have been injected intravenously in Balb-c mice at different time points following immunization with tetanus toxoid (TT). Thus, we analyzed at different time points the TT specific serum antibody titres and TT specific T cell proliferative response from the peripheral blood of Balb-c mice. Upon sacrifice, the frequency of antigen specific plasma cells, memory B cells and T cells have been analyzed from the bone marrow, lymph nodes and spleen. In the latter, the extent of germinal centres formation has been evaluated.Overall we show that in vivo administration of MSC affect both T and B cell response to a non self antigen such as TT but does not lead to a condition of immunodeficiency. These results are of pivotal importance due to the possible utilization of MSC for the treatment of autoimmune diseases such as multiple sclerosis. We refolded bacterially produced HuMOG and compared pathogenicity of refolded HuMOG versus non-refolded HuMOG in DA rats. Whereas DA rats immunized with non-refolded HuMOG remained perfectly healthy, DA rats immunized with refolded HuMOG developed severe disease. No differences in MOG-directed T-cell responses could be observed between both groups. In contrast, MOG serum antibodies from both groups differed in their capacity of binding native MOG expressed on the surface of eukaryotic cells as measured by FACS. These data underscore the importance of using correctly refolded HuMOG for studying MOG antibody responses in MS patients. The precise role of B cells and humoral immunity in both Multiple Sclerosis (MS) as well as Experimental Autoimmune Encephalomyelitis (EAE) remains a subject of intense debate. While intrathecal immunoglobulin (Ig) production and Ig-deposition in inflammatory lesions is a hallmark of MS, mice deficient in B cells and Igs have been shown to develop severe EAE. On the other hand, mice deficient in FcRã, the signaling entity of activating Fc-receptors, are resistant to EAE-induction. To resolve this apparent paradox we generated mice deficient in both B cells and FcRã and induced EAE through immunization with both recombinant MOG 1-121 protein or MOG 35-55 peptide. The functional expression of FcRã on systemic accessory cells, but not CNS-resident cells, appears to be vital for the development of CNS-inflammation independent of lymphocytes. While these data clearly dismiss any involvement of B cells as mediators in EAE, we found that the injection of Abs directed against MOG drastically worsens EAE severity, inflammation and demyelination in MOG induced EAE. We conclude that Abs generated during the course of MOG-peptide orprotein induced EAE are irrelevant for the pathogenesis of EAE, but that under certain conditions Abs are capable of driving demyelination in an FcRã-independent but complement-dependent fashion. PP06-03 Induction of adaptive regulatory T cells during recovery of EAE sensitized with PLP136-150 in SJL/J mice: The presence of suppressor epitope within PLP Youwei Lin, Sachiko Miyake, Takashi Yamamura Department of Immunology, National Institute of Neuroscience, NCNP, Japan SJL/J mice are known to develop chronic, relapsing EAE with epitope spreading after immunization with PLP139-151. Although genetic factors were employed to account for the relapsing-remitting course of this EAE, we found that immunization of SJL/J mice with an overlapping peptide PLP136-150 induced monophasic EAE without relapse, indicating that peptide sequence used for primary immunization is critical for determining the clinical course. Whereas both immunodominant PLP peptides induced cross-reactive T cells, immunization with PLP136-150 tended to generate Th2-biased population, as compared to PLP139-151. More strikingly, mice in the recovery phase of PLP136-150-EAE were completely resistant against re-induction of EAE; whereas PLP139-151-EAE mice were fully susceptible to EAE re-induction. Furthermore, we found that after recovery from PLP136-150-induced EAE, suppressor cells would emerge in the draining lymph nodes (LN) that could confer protection against EAE upon cell transfer. These suppressive LN cell populations contained a higher number of adaptive regulatory CD4 + CD25 + T cells (Treg) that would co-express CD103 and CD69. Adoptive transfer experiment showed that CD103 + CD69 + Treg cells induced by PLP136-150 immunization have the highest potency to suppress EAE in vivo. Our results indicate that the benign nature of PLP136-150 induced EAE may result from the induction of strongly suppressive CD103 + CD69 + Treg cells. (Abbreviations: EAE = experimental autoimmune encephalomyelitis, PLP = proteolipid protein) PP06-04 Paralysis of CD4 + CD25 + regulatory T cell response in chronic autoimmune encephalomyelitis Yoh Matsumoto, Hiroshi Sakuma, Kuniko Kohyama, Mie Nakajima, Il-Kwon Park Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan To elucidate the pathomechanisms of relapse and chronicity of multiple sclerosis, we examined the role of CD4 + CD25 + regulatory T cells (Treg) in its animal model, chronic experimental autoimmune encephalomyelitis (EAE). Chronic EAE was induced in LEW.1AV1 rats by immunization with myelin oligodendrocyte glycoprotein (MOG) and the results obtained were compared with those from acute EAE. In chronic EAE, CD25 + T cells in EAE lesions in the central nervous system (CNS) decreased rapidly at the beginning of the disease, while these cells were maintained at high levels during the recovery from acute EAE. Consistent with this finding, the levels of Foxp3 mRNA in the CNS was significantly lower throughout the course of chronic EAE than that at the recovery of acute EAE. The number of Foxp3 + CD4 + CD25 + Treg and the levels of Foxp3 mRNA in the lymphoid organ were also significantly lower in chronic EAE. However, there was no difference in the regulatory function of individual CD4 + CD25 + Treg cells isolated during the disease course between acute and chronic EAE. Furthermore, the adoptive transfer of CD4 + CD25 + Treg that had been activated with anti-CD28 mAb (JJ316) delayed the onset of chronic EAE (after one transfer) and suppressed the development of the disease completely (after two transfers). These findings suggest that impairment of the CD4 + CD25 + Treg response, but not the function of individual cells, is critical for the development of chronic autoimmune diseases and can be adjustable by autologous Treg transplantation. PP06-05 Natural killer (NK) cells exert cytotoxic effect on autoantigenspecific, encephalitogenic T cells in experimental autoimmune encephalomyelitis The mechanism of natural killer (NK) cell regulatory role in experimental autoimmune encephalomyelitis (EAE) was studied in SJL/J mice. In vivo experiments showed that NK cell depletion by anti-NK1.1 monoclonal antibody treatment enhanced EAE in mice. To investigate the mechanism, we cultured proteolipid protein (PLP) 136-150 peptide-specific, encephalitogenic T cell lines, which were used as the NK cell target. Our results show that NK cells exert a direct cytotoxic effect on autoantigenspecific, encephalitogenic T cells. Furthermore, cytotoxicity to PLPspecific, PP06-06 Encephalitogenic CNS dendritic cells drive the activation of naïve self-reactive CD4 + T cells Our recent findings show that CD11c + DCs, but not macrophages (Mϕ) or microglia isolated from the CNS of SJL mice with proteolipid (PLP)178-191 induced relapsing experimental autoimmune encephalomyelitis (R-EAE), induced the proliferation of naive (CD62L hi ) transgenic CD4 + T cells specific for the immunodominant myelin peptide PLP139-151 (139TCR) with antigens collected in situ. By laser sorting CNS DC subtypes to high purity at the peak acute phase of R-EAE, we demonstrate CD11b + DCs induce 139TCR expansion 44 fold, CD11b − B220 − CD8α + DCs (DNDC) and CD11b − B220 + DCs induce 6 and 5 fold expansion, respectively, and Mϕ 2 fold expansion without peptide being added to the cultures. CNS DC driven 139TCR proliferation is MHC class II and B7-1/B7-2 dependant. Levels of IFNγ and IL-2 secreted by 139TCR cells are highest with CD11b + DCs and DNDCs as activators. 139TCR cells closely associate with CD11b + DCs in CNS inflammatory foci in SJL:139TCR mixed bone marrow chimera (BMC) mice during peak acute R-EAE. The 4 CNS APCs internalize PLP in the CNS. The abundance of DCs in the CNS correlates strongly with EAE severity. These data imply that discrete DC populations are recruited to the inflamed CNS that have distinct roles for driving naïve T cell activation in that organ. This work is supported by grants from the National Multiple Sclerosis Society (USA) and the National Institutes of Health. PP06-07 Traffic of CSF-circulating dendritic cells during experimental auto-immune encephalomyelitis It is now established that dendritic cells (DCs) are able to migrate from the CNS to the cervical lymph nodes, through a yet unidentified pathway. We showed that DCs injected into the CSF of rats target the B-cell follicles of cervical lymph nodes (1) . Here, we explored the migratory behavior of CSFcirculating DCs under neuroinflammatory conditions. For this purpose, bone marrow-derived myeloid DCs were labeled with a fluorescent marker (CFSE) then injected stereotaxically into the lateral ventricles of EAE rats. Rats were sacrificed on day 1 or 8 following injections and brains, cervical lymph nodes and axillary lymph nodes were assessed for the presence of CFSE-labeled cells. In addition, intraventricular injections of fluorescent microspheres were performed in order to track endogenous CSF-circulating antigen presenting cells. We found that in the CNS, injected DCs localized in meninges, velums, Virchow-Robin spaces and in several periventricular areas. Similar results were obtained when tracking endogenous MHC class II + cells with fluorescent microspheres. In the lymph nodes, CFSE-labeled DCs were evidenced in the B-cell zones of cervical but not axillary lymph nodes. Similarly, cells having engulfed fluorescent microspheres were detected in the B-cell zone of cervical but not axillary lymph nodes. Finally, using Western blot analysis, we observed that IgG recovered from cervical versus axillary lymph nodes differ qualitatively and quantitatively in their ability to recognize CNS antigens. Altogether, these results suggest that during EAE, CSF-circulating DCs may be responsible for the development of a preponderant B-cell response, which takes place within the cervical lymph nodes. (1) Hatterer E., Davoust N., Didier Bazès M., Vuaillat C., Malcus C., Belin M.F. and Nataf S. How to drain without lymphatics? Dendritic cells migrate from the cerebrospinal fluid to the B-cell follicules of cervical lymph nodes. PP06-09 Vaccination-induced autoimmunity in the absence of secondary lymphoid tissues M. Greter, P. Bargsten, B. Becher Institute of Neurology, Department of Neuroimmunology, University Hospital Zurich, Switzerland Subcutaneous (s.c.) vaccination delivers antigen (Ag) to local dendritic cells (DCs) which subsequently migrate through afferent lymphatics into draining lymph nodes (LNs) where they encounter lymphocytes recognizing their cognate Ag to initiate immunity. Experimental autoimmune encephalomyelitis (EAE) serves as the animal model for multiple sclerosis, a demyelinating autoimmune disease of the central nervous system (CNS). Secondary lymphoid tissues are widely held to be vital for the initiation of adaptive immunity. Here we demonstrate that alymphoplasia (aly) mice, which are characterized by the complete lack of lymph nodes (LNs) and peyer's patches, are resistant to EAE induced by s.c. immunization with MOG. However, the immunodeficiency results from the impact the aly lesion has on immunity but not from the lack of lymphoreticular structures. This led to the exciting understanding that vaccination induced autoimmunity of the CNS can develop autonomously and without dedicated immune structures. We clearly show that while B cell activation requires a lymphoreticular system and germinal center formation, T cell immunity functions independent of secondary lymphoid tissues. We demonstrate that in the absence of secondary lymphoid tissues the liver acts as a site where primary immunity can occur. Inflammation induced tertiary follicles are well described. However this is the first report indicating that vaccination induced fulminant autoimmunity initiated in the liver. PP06-10 Role of innate immunity in autoimmune disease: Toll-Like Receptor agonists can trigger autoimmune encephalomyelitis Infectious organisms may play a role in human autoimmune diseases. Experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS), is induced in Lewis rats with rat myelin basic protein (MBP) peptide MBP68-86 in complete Freund's adjuvant (CFA). Although the role of mycobacteria is unclear, it is required for the induction of EAE. To investigate whether Toll-like receptor (TLR) agonists can replace mycobacteria in adjuvant, we immunized rats with MBP68-86 in incomplete adjuvant (lacking mycobacteria), but supplemented with TLR agonists. Rats immunized with either 100 μg MBP68-86 plus 100-300 μg cytosineguanine dinucleotide-containing oligodeoxynucleotide (CpG), a TLR9 agonist, or MBP68-86 plus 50 μg lipopolysaccharide (LPS), a TLR4 agonist, did not develop EAE, although their T cells proliferated and secreted interferon-gamma when stimulated with MBP68-86. Spleen cells proliferated and secreted interferongamma in response to MBP68-86, and secreted IL-12 in response to CpG or LPS, but not to MBP68-86, suggesting that the TLR agonists promote a Th1 response. Thus, a combination of CpG and LPS can substitute for mycobacteria for induction of EAE. We conclude that a combination of TLR agonists, in the presence of autoantigen, can trigger autoimmune disease via the innate immune system. PP06-11 The innate immune response to adjuvants dictates the adaptive immune response to autoantigens M. Staykova, D. Linares, S. Fordham, G. Bartell, W. Cowden, D. Willenborg Neurosciences Research Unit, ANU Medical School, Canberra Hospital, Canberra, Australia DA and Lew rats are known as "susceptible", PVG ratsas "semisusceptible" (30% of the females develop EAE) and BN ratsas "resistant", based on the development of EAE after immunisation with myelin basic protein or spinal cord homogenate emulsified in complete Freund's adjuvant (MBP-CFA, SCH-CFA). We re-examined EAE susceptibility/resistance in these rat strains based on our knowledge that nitric oxide (NO) can affect generation of T effector cells and/or their transendothelial migratory properties. If another adjuvant is usedcarbonyl iron (CI) which does not increase the systemic NO levelsall four rat strains develop acute EAE. There were no significant differences in lymph node and spleen percentages of T lymphocytes (CD4 + and CD8 + ), B lymphocytes and NK cells when SCH-CFA or SCH-CI were used. There was a significant difference between (DA and Lew) and (PVG and BN) in the macrophage/monocyte numbers, expression of CD80 and CD86 costimulatory molecules and iNOS activity. The cellular composition of CNS inflammatory infiltrates was also different: in DA and Lew the majority of the cells were lymphocytes with some ED1 + cells while in PVG and BN there was massive ED1 positivity. Experiments with rat chimeras and Indian ink loaded monocytes showed that the ED1 + cells in DA and Lew were mainly macrophages while in PVG and BN there was massive glial cell activation. PP06-12 Blockade of TREM-2 exacerbates experimental autoimmune encephalitis Little is known about the function of DAP12/TREM-2 in the central nervous system (CNS). However, a rare autosomal recessive condition, Nasu-Hakola disease (NHD) is associated with loss-of-function mutations in DAP12 and TREM-2. The brain pathology observed in NHD patients suggests that disruption of the TREM-2/DAP12 pathway leads to neurodegeneration with demyelination and axonal loss. The AIM of this study was to determine the role of TREM-2 in experimental autoimmune encephalomyelitis (EAE). Methods: TREM-2 protein expression was investigated using a newly produced monoclonal antibody (MAb) against the mouse TREM-2 receptor. Blocking effect of anti-mouse-TREM-2 MAb was assessed using BWZ.36-TREM-2 cell reporter system. Results: TREM-2 protein expression was demonstrated in vitro on macrophages, microglia and dendritic cells. We report that TREM-2 was up-regulated in the spinal cord during both the early inflammatory and chronic phases of EAE. We also demonstrate that TREM-2 was highly expressed on microglial cells in the CNS during EAE. Blockade of TREM-2, obtained treating the mice with the anti-TREM-2 MAb in the effector phase of EAE, results in disease exacerbation with more diffuse CNS inflammatory infiltrates and demyelination in the spinal cord. Conclusions: Our data show a critical role for TREM-2 during inflammatory responses in the CNS and suggest that TREM-2 plays a role in the pathogenesis of autoimmune inflammatory/demyelinating diseases of the CNS. PP06-13 Type 1 interferon receptor (IFNAR)-dependent modulation of myeloid cell activation determines the course of experimental autoimmune encephalomyelitis While treatment of MS patients with interferon-b (IFN-b) leads to a marked decrease in the exacerbation rate as well as to delayed sustained disease progression, the precise mechanisms of the beneficial effects of IFNβ are still enigmatic. In this study we show that type 1 interferon receptordeficient mice (IFNAR −/− ) were highly susceptible to experimental autoimmune disease (EAE) and developed a more severe disease course with increased CNS inflammation, demyelination and lethality. To clarify this, we used Cre/loxP-mediated gene targeting to investigate the cell-specific function of IFNAR in vivo. Mice with a specific IFNAR deletion in the central nervous system (Nestin-CreIFNARflox/flox) revealed no differences in the clinical course and showed compatible tissue damage in the CNS compared to WT controls, indicating that type 1 interferons do not have a direct protective impact on the CNS. Interestingly, neither T cell (CD4 − CreIFNARflox/flox) nor B cell (CD19 − CreIFNARflox/flox)-specific IFNAR deletion influenced the clinical course and cellular composition of infiltrating cells. However, IFNAR deletion on macrophages/neutrophils (LysM-CreIFNARflox/flox) led to severe disease with an enhanced effector phase and increased disease lethality as seen in IFNAR −/− mice. Deletion of IFNAR in macrophages induced altered MHC class II expression and change of cytokine and chemokine production. In summary, we show that IFNAR triggering, specifically on myeloid cells, but not lymphocytes or CNS cells, is crucial for immunomodulatory effects of type 1 IFN during autoimmune CNS disease. PP06-14 The effects of fibroblast growth factors 1 and 2 on Experimental Autoimmune Encephalomyelitis (EAE) Objective: To investigate the roles of FGF1 and FGF2 in EAE progression. Background: FGFs are potent mitogens and glial cell activators that exhibit broad-spectrum neurotrophic activity. However, it is not known how FGF may affect EAE progression and cytokine profiles in EAE. Design/methods: C57BL6 wt, FGF1/2 double knockout and FGF1/2 heterozygote mice were immunized with MOG 35-55 emulsified in CFA. Clinical scores were assessed daily. Serum was taken from day 21 animals immediately preceding euthanasia. Multiplex assays were performed to determine serum cytokine levels in knockouts and heterozygotes. Results: FGF-1/-2 knockouts developed significantly milder clinical symptoms compared to heterozygote and wt mice. Serum levels of IL4, IL10, and KC (CXCL1) levels were significantly higher in FGF1/2 knockout than those in heterozygote mice. Other cytokines including IL1, IL2, IL3, IL5, IL6, GM-CSF, IFN-γ, TNF-α, IL12, IL17, G-CSF, MIP-1α, and RANTES levels showed no significant differences. Conclusions: FGF1/2 knockout mice developed milder EAE than heterozygotes and wt mice, which correlates with serum anti-inflammatory cytokine (TH2) levels. Our findings suggest that Th2 cells are up regulated in the absence of FGF1 and FGF2. Gene expression levels for IL4, IL10, and other cytokines in the CNS are currently being measured. PP06-15 Neurotrophic factors and neuroinflammation: Experimental autoimmune encephalomyelitis in conditional BDNF-knock out mice Brain derived neurotrophic factor (BDNF) is involved in neuronal and glial development and survival. It is mainly produced by neurons, but also by leukocytes in vitro and in multiple sclerosis (MS) lesions. Yet, the functional relevance of BDNF expression by immune cells in autoimmune demyelination is still unknown, since conventional BDNF knockout mice die prematurely. We applied the Cre/loxP system to generate mice with a conditional deletion of BDNF in the T-cell lineage (lckCre BDNF flox/− mice) or in myeloid cells (lysMCre BDNF flox/− mice). In these mice, experimental autoimmune encephalomyeltits (EAE) was actively induced with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55.Compared to control mice, MOG-EAE in lckCre BDNF flox/− mice is more severe (mean score on day 43 p.i. Histological analyses during the chronic disease phase revealed an increased inflammatory infiltration in lckCre BDNF flox/− mice. MOG 35-55 EAE in lysMCre BDNF flox/− mice was of similar severity in the chronic phase of the disease. The immune reaction in lysMCre BDNF flox/− mice was attenuated with a decrease in interferon-gamma production and reduced inflammatory infiltration by at least 60%. Despite less inflammation in chronic MOG-EAE, the extent of axonal damage was not different between lysMCre BDNF flox/− mice and controls. Our findings suggest a novel role of immune-cell derived BDNF in immunoregulation, but also underscore its importance as susceptibility factor for maintaining axonal integrity. These data further support the concept of neuroprotective autoimmunity.Supported by SFB 581 TPA1. Multiple sclerosis (MS) is an autoimmune, demyelinating disease of the central nervous system. EAE is a widely used animal model of MS. EAE can follow either a relapsing-remitting (RR) or a chronic (CH) disease course. To date, the molecular and pathological differences that underlie these different forms of MS and EAE are not fully understood. Expression of SOCS-1 and SOCS-3 mRNA increases in the early stages of RR disease and decreases during the subsequent remission phase. In contrast, SOCS-1 and SOCS-3 mRNA expression is elevated at all stages of CH disease. As the expression of SOCS is turned on by pro-inflammatory cytokine signaling, these data suggest that the inflammatory response is prolonged in the CH form of EAE. Expression of SOCS-1 was localized to immune cells within EAE lesions, while SOCS-3 was predominantly expressed in glial cells adjacent to lesions. These results demonstrate that SOCS-1 and SOCS-3 are preferentially upregulated in distinct cell types during the course of EAE. PP06-17 Regulation of sonic hedgehog mediated neural stem cell differentiation in experimental autoimmune encephalomyelitis Yue Wang, Jaime Imitola, Stine Rasmussen and Samia J. Khoury Center for Neurologic Diseases, Harvard Medical School, Boston, USA Objectives: To investigate the role of sonic hedgehog (Shh) signaling in neural stem cell (NSC) and precursor cell differentiation, and the effect of inflammation during experimental autoimmune encephalomyelitis (EAE) on this signaling. Methods: Expression of Shh, its receptor smoothened (SMO), and the transcription factor Gli1 was examined in the spinal cords of C57BL/6 mice with MOG-induced EAE by immunohistology; IFN-γ-treated NSCs were examined by real-time PCR and Western blot for Shh and Shh-induced Gli1 expression; effect of Shh on astrocyte-mediated NSCs differentiation was evaluated by anti-Shh antibody or control IgG treated NSCsadult spinal cord astrocytes co-culture; IFN-γ-treated and untreated NSCs were compared for Shh-mediated differentiation in vitro. Results: Reactive spinal cord astrocytes upregulated Shh and SMO in EAE, especially around perivascular infiltrates and areas of demyelination, while Gli1 was downregulated. NSCs differentiated into neurons, astrocytes and oligodendrocytes when co-cultured with adult astrocytes, but anti-Shh significantly decreased the number and the maturity of neurons; Shhmediated NSC differentiation was significantly inhibited by IFN-γ, which was independent of cell death. Conclusions: Shh is an important mediator for adult astrocyte-induced NSC differentiation and it is upregulated in reactive astrocytes during EAE. However, IFN-γ may play a critical role in inhibiting Shh-mediated NSC and precursor cell differentiation by downregulating the Shh signaling molecule Gli1 in EAE. PP06-18 Endogenous prion protein regulates the inflammatory response and the extent of CNS damage in a murine model of multiple sclerosis S. Tsutsui, F. R. Jirik Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB Canada T2N 4N1 The role of PrP C in the pathogenesis of neuroinflammatory and neurodegenerative diseases remains elusive. However, a significant number of in vitro experiments have provided evidence that PrP C is neuroprotective, and moreover, PrP C is expressed on lymphocytes, macrophages and myeloid dendritic cells, suggesting that PrP C might potentially be playing a critical role in neuroinflammatory and neurodegenerative diseases. To determine the effects of reduced PrP C expression in a model of MS, experimental allergic encephalomyelitis (EAE), homozygous PrP C knockout (PrP −/− ) mice and littermate wild type (PrP +/+ ) controls were treated with subcutaneous myelin oligodendrocyte glycoprotein (MOG 35-55 ) to induce EAE. Animals were monitored for clinical signs and graded daily. Brain and spinal cord sections from euthanized animals were stained with H-E and silver. PrP −/− mice exhibited an earlier onset and a significantly more severe EAE phenotype than PrP +/+ controls in neurobehavioral score. During the EAE, H-E staining showed marked perivascular infiltration in brain. Silver staining showed the number of axons was reduced in spinal cord of PrP −/− mice compared to PrP +/+ controls. These studies suggest that lack of PrP C expression dysregulates the EAE inflammatory response, and might also render CNS cells more susceptible to injury during. Elucidating the role of PrP C in EAE will lead not only to a better understanding of neurodegenerative disease pathogenesis but also raises the possibility that alterations in prion protein levels or function might be of therapeutic benefit. PP06-19 Critical role for CD5-dependent CK2 activation signals in EAE: Attenuated disease is associated with diminished numbers of γIFN + IL17 + T H -cells in the CNS Chander Raman 1 , Robert C. Axtell 1,2 and Scott R. Barnum 2 Departments of Medicine 1 and Microbiology 2 , University of Alabama at Birmingham, Birmingham, AL 35294, USAWe recently reported that mice lacking CD5, a negative regulator of Tcell activation, unexpectedly exhibited delayed onset and decreased severity to MOG-peptide induced experimental autoimmune encephalomyelitis (EAE). The attenuated disease was a consequence of decreased ability of activated CD5 −/− T-cells to persist. This led to the discovery that CD5 had an engagement-dependent prosurvival activity in T-cells. To test the prediction that the prosurvival activity was mediated by CK2, an anti-apoptotic serine/ threonine kinase that constitutively associates with CD5, we generated mice that expressed CD5 unable bind/activate CK2 using transgenic and "knockin" approaches. Remarkably these CD5-CK2 binding/activation-deficient mice were very resistant to development of EAE. Resistance was not associated with absence of T-cell infiltration in the CNS but rather due to decrease in T H -cells that coexpress both γIFN and IL-17. The newly described T H 17 population of T-cells developed with equal efficiency in WT and CK2-binding/activation deficient CD5 mice. We further show that Tcells deficient in CD5-CK2 signaling hyperproliferate following primary stimulation, however, respond very poorly to restimulation compared to WT-CD5 T-cells. Our results indicate that the T H IFNγ-IL17 is likely to be an important pathogenic effector population in multiple sclerosis. We also show that the CD5-CK2 pathway is an attenuator of T-cell activation and cell death. The coordinate effect of these activities of CD5 is likely to be important in differentiation and persistence of effector T-cells in autoimmunity and immunity to pathogens. NF-kB activation and phosphorylation of IkB is associated with development and progression of the experimental autoimmune encephalomyelitis Insun Hwang, Taekyun Shin, Youngheun Jee Department of Veterinary Medicine, Applied Radiological Science Institute, Cheju National University, Jeju, 690-756, South Korea Development of autoimmune disease requires coordinated expression of a number of immune-related genes which mediate many inflammatory responses. A key player in regulation of inflammatory gene expression is the nuclear factor-kB (NF-kB) activated by phosphorylation and proteolytic degradation of inhibitory protein IkB. However, involvement of NF-kB of transcription factor in various immune-mediated diseases has been proposed but the direct supporting evidence is still lacking. To elucidate the roles of NF-kB and phospho-IkB in the development and progression of EAE, we investigated the expression of nuclear NF-kB (p65) and phospho-IkB in the central nervous system (CNS) of rats during experimental autoimmune encephalomyelitis (EAE). In Western blot analysis, phospho-IkB expressed rapidly at the early stage and increased significantly at the peak stage of disease. In addition, the expression of NF-kB increased at the early and peak stage followed by slight decline at the later stages. The expression of nuclear NF-kB and phospho-IkB was parallel to severity of EAE. Immunohistochemical studies showed that the NF-kB and phospho-IkB immunoreactivity was mainly expressed in inflammatory cells (macrophages, T cells) and glial cells (astrocytes, microglial cells) at the peak stage and disappeared at the recovery stage of disease.These findings suggest that the activation of NF-kB and phosphorylation of IkB is closely associated with autoimmune inflammation in the CNS and plays an important role in the initiation and progression of EAE. PP06-21 Anti-S-nitrosocysteine antibodies are not triggered by nitric oxide production A.I. Boullerne, A. FeinsteinUniversity of Illinois at Chicago, Chicago, Illinois, USA An autoimmune antibody response against nitrosylated cysteine (SNOcysteine-BSA) was found elevated in plasma of multiple sclerosis patients and correlated with clinical activity (Boullerne et al. 22:123) , suggesting that production of nitric oxide (NO) triggered an autoimmune response. To test this hypothesis, mice immunized to develop experimental allergic encephalomyelitis (EAE) were monitored for antibody responses and NO production in plasma. Wild-type C57Bl/6 and NOS-2 null mice were immunized with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG) followed a week later by a MOG booster and treatment with anti-inflammatory PPAR-gamma agonists. The strong increase of NO observed a week after immunization was lessened in low-dose treated mice and abolished in high-dose treated mice with no clinical signs. An increased antibody response was found two weeks after immunization in correlation with clinical signs arising a week later. In the absence of clinical signs there was no antibody response. NOS-2 null mice with EAE did not show increases in NO production over a month follow-up. Their antibody response was reduced by half and showed an opposite fluctuation compared to wild-types: treatment exacerbated the antibody response while clinical signs decreased it. These findings point to a complex role of PPAR-gamma agonists that may promote a Th2-driven antibody response in the absence of NO production which could explain the presence of an antibody response in NOS-2 null mice. PP06-22 Increased phosphorylation of caveolin-1 and p38 mitogenactivated protein kinase in EAE The expression of phospho-specific caveolin-1 (p-caveolin-1) and phosphorylated p38 mitogen-activated protein kinase (p-p38) was analyzed in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis (EAE). Western blot analysis showed that p-caveolin-1 and p-p38 were constitutively expressed in normal spinal cords and that it significantly increased in the spinal cord with EAE at peak stages of EAE (P < 0.05), and decreased slightly at the recovery stage of EAE. Immunohistochemistry showed that p-caveolin-1 was constitutively expressed in few vascular endothelial cells and glial cells of the normal rat spinal cord. In EAE lesions, p-caveolin-1 was intensely immunostained in inflammatory T cells and macrophages. p-p38 was constitutively expressed in neurons, glial cells, and vascular endothelial cells in normal spinal cords and was also expressed in inflammatory cells, as well as increased expression in glial cells in EAE. Taken all into considerations, we postulate that the phosphorylation of caveolin-1 and p38 play an important role in the pathogenesis of EAE. PP06-23 Activation of mitogen-activated protein kinases in experimental autoimmune encephalomyelitis T. Shin Department of Veterinary Medicine, Cheju National University, Jeju, Republic of Korea Increasing evidence suggests that mitogen-activated protein (MAP) kinases, including extracellular signal-regulated kinase (ERK), c-Jun NH(2)terminal protein kinase (JNK), and p38, are involved in the pathogenesis of human demyelinating diseases and its animal model experimental autoimmune encephalomyelitis (EAE). To assess the involvement of MAP kinases in EAE, the expression and localization of phosphorylated form of ERK(p-ERK), JNK(p-JNK) and p38(p-p38) was analyzed in EAE in rats. Western blot analysis showed that all MAP kinases including p-ERK, p-JNK, and p-p38 were significantly increased in the EAE lesions at the peak stage (p < 0.05), and slightly declined in the recovery stage. As well, both MAPK/ ERK kinase 1, an upstream activator, and cAMP responsive element binding protein (CREB), a downstream transcription factor, was activated in EAE affected spinal cords. Immunohistochemistry showed that p-ERK was constitutively expressed in brain cells, including astroglial cells, and showed enhanced immunoreactivity in those cells in EAE, while some T cells and macrophages were positive for p-ERK in EAE lesions. Both p-JNK and p-p38 were intensely immunostained in T cells in EAE lesions. The majority of brain cells and inflammatory cells were positive for p-CREB in EAE lesions. These findings suggest that activation of MAP kinases and its downstream transcription factor play an important role in the initiation of EAE paralysis and subsequent recovery. PP06-24 Deletion of Pten in antigen-activated T lymphocytes reveals a potential role for Pten in effector populations in the experimental autoimmune encephalomyelitis (EAE) disease model Department of Biochemistry and Molecular Biology, University of Calgary, Calgary AB, Canada Pten, a phosphoinositol phosphatase, is critical to T lymphocyte ontogeny and self-tolerance via the negative regulation of pathways lying downstream of phosphoinositide 3′ kinase (PI3K). Previous studies using the Cre-loxP system to delete Pten genes in developing thymocytes, have revealed defects in both thymic selection and T cell homeostasis. To examine the functional role of Pten specifically in mature effector T lymphocytes, and in the context of a normal pre-immune T-cell repertoire, we selected a system that allows Cre-mediated gene excisions to occur within CD8 + and CD4 + T cells subsequent to activation. This system, which employs the Granzyme B gene promoter to drive expression of the Cre recombinase upon TCR activation, was used to trigger excisions of exons 4 and 5 of the floxed Pten gene. In mice homozygous for the floxed allele, we were thus able to obtain Pten deletion specifically in a subset of activated T lymphocytes. To characterize this system further, we evaluated the efficiency of Pten gene deletion in mature T cells in vitro, and also studied the consequences of Pten loss in a model of T cell-mediated immunopathology, murine experimental autoimmune encephalomyelitis (EAE). EAE was induced by injection of a synthetic myelin oligodendrocyte glycoprotein peptide (MOG 35-55) in complete Freund's adjuvant and Pertussis toxin. Initial clinical and neuropathological characterization of the MOG-immunized Granzyme B Cre x floxed Pten mice indicates that Pten plays a critical role in regulating not only the severity of EAE, but also the time-course of this disease. PP06-25 Exacerbation of experimental autoimmune encephalomyelitis in P2X7R −/− mice Lanfen Chen and Celia F. Brosnan Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, 10461, USA P2X 7 receptor (P2X 7 R), an ATP-gated ionotropic receptor, has been proposed to serve as a regulator of inflammation. In this study we tested whether the development of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, was modified in animals lacking the P2X 7 R. In P2X 7 R −/− mice clinical and pathological signs of EAE were more severe than in WT mice. Bone marrow radiation chimeras revealed that greater susceptibility to EAE was detected in chimeric mice of WT host engrafted with P2X 7 R −/− BM cells, indicating that the genotype of the BM cells regulated disease susceptibility. In vitro, spleen and lymph node cells from P2X 7 R −/− mice had a higher proliferative activity and less apoptosis in response to MOG peptide. Similarly, significantly fewer Annexin V/PI + and TUNEL positive lymphocytes were found in the CNS of P2X 7 R −/− mice early in the disease. Cytokine profiles of the culture supernatants showed significantly decreased expression of IFNγ and IL-6 in MOG-activated spleen cells from P2X 7 R −/− mice. QPCR and protein assays showed that protein for IL-1β (but not mRNA), as well as mRNA and protein for IL-6 and IFNγ were significantly reduced in spinal cord homogenates. From these data we conclude that the enhanced susceptibility of P2X 7 R −/− mice to EAE reflects a more vigorous proliferative response to MOG, lower levels of IFNγ, nitric oxide and IDO compared to WT mice, which correlated with loss of apoptotic activity in lymphocytes. PP06-26 Macrophage phenotype during relapsing experimental autoimmune encephalomyelitis can be predicted by MRI at the disease onset EA2966 Neurobiology of Myelin Disorders Laboratory, University Victor Segalen, Bordeaux, France Macrophage infiltrates in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS) can be monitored in vivo by MRI with ultrasmall super paramagnetic iron oxide (USPIO) nanoparticles. In pathological conditions macrophages present at least two main phenotypes: proinflammatory M1 macrophages express inducible Nitric Oxide Synthase (iNOS) while immunomodulatory M2 macrophages express Arginase I.We used a relapsing EAE to study macrophage phenotypes within inflammatory infiltrates at the clinical onset (group I), at the end of 1st attack (group II) and during the 2nd attack (group III). USPIO MRI was performed at the clinical onset and was repeated for animals of group III. Animals with MRI signal changes were considered as MRI + . On serial thin sections of brainstem activated macrophages were quantified by anti ED1, M1 macrophages by anti iNOS labelling using confocal immunofluorescence and M2 by arginase I labelling; axonal damage and loss in brainstem tissue were quantified by classic immunohistochemistry methods. The obtained results were compared with MRI data.MRI + rats presented more important macrophage recruitment (ED1 + cell number); cells expressing ED1 + and iNOS + (M1) were also more abundant in MRI + animals than in MRI − animals. In regard to clinical disease severity and tissue alterations (axonal loss and tissue damage) significant differences between MRI + and MRI − animals were observed. M2 phenotype analysis is in progress.In conclusion, MRI performed at the onset of EAE predicts the presence of iNOS-expressing macrophages (M1 phenotype) and the extend of nervous tissue lesions in inflamed CNS at different stages of disease course. Magnetic resonance imaging (MRI) techniques are widely used to investigate multiple sclerosis (MS) lesions. Histopathological correlates of MRI signal alterations are still poorly defined. Here, we compared MRI and histopathology in a murine experimental autoimmune encephalomyelitis (EAE) model. Brainstem lesions were induced by the adoptive transfer of a PLP139-51 specific T-cell clone in SJL/J mice. 25 of 27 histopathologically identified acute EAE lesions were retrieved by T1and T2-weighted high-resolution 3D MRI. Two dominant MRI lesion patterns could be identified: Lesions with reduced signal intensity on T1-and T2-weighted MRI (type A) and lesions with isointense or slightly reduced signal intensity on T1-weighted MRI and increased signal intensity on T2-weighted MRI (type B). Type A lesions were characterized by significantly denser inflammatory cell infiltrations and more myelin and axonal loss than type B lesions. Comparative analysis revealed that lesional cellularity, myelin loss and axonal density correlated with signal intensities obtained by T1-and T2-weighted images in the lesion center. In perilesional areas, microglia cell numbers and Ig deposition correlated strongly with signal intensities on T2-weighted MRI. Gd-DTPA enhancement correlated with Ig deposition and mainly took place in areas with activated microglia cells. Given the robust correlation between MR signal intensities and histopathological changes the model can be used for MRI evaluation of new therapeutic strategies. PP06-28 Magnetic resonance-guided evaluation of disease burden in mice with chronic experimental autoimmune encephalomyelitis (EAE) Magnetic resonance (MR) may help pre-clinical studies in monitoring pathological abnormalities of brain white matter. We investigated whether inflammatory demyelinating lesions affecting the brain of mice with experimental multiple sclerosis (MS) may be detected and monitored over the natural story of the disease model with a 3T human MR apparatus. Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice using 200 μg/mouse of myelin-oligodendrocyte glycoprotein (MOG)35-55 in complete Freund's adjuvant, while sex-, strain-, weightand age-matched both non-immunized and CFA-immunized mice were used as controls. Mice (n = 5/group) were evaluated for both basal as well as gadolinium (Gd) contrast enhanced MR images at pre-clinical, acute-and chronic phases of the disease (e.g., 10, 15, and 32 days after immunization [dpi], respectively). Meningeal enhancement and T1 Gd enhancing parenchymal lesions started appearing as early as from 10 days dpi, while peak of "active" demyelinating lesions were recorded at clinical onset (17 dpi). Meningeal enhancement faded over time, progressively disappearing at the 25 dpi. In 50% of EAE mice, new enhancing lesions were detected at 32 dpi, thus indicating a sub-clinical relapse. 74% of examined mice presented at least one demyelinating lesion in the cerebellum, 64% of them had lesions in either the corpus callosum or the external capsule. Neuropathological analysis confirmed the presence of lympho/monocyte infiltrates in meninges and parenchymal T1-weighted Gd-enhanced images. Further studies will be necessary to more carefully evaluate sensibility and specificity of human graded MR in detecting acute and chronic demyelination in EAE mice. Infiltration of monocytes into the central nervous system is a key event in neuroinflammatory diseases. The development of superparamagnetic particles of iron oxide (SPIO) has made magnetic resonance imaging (MRI) a valuable tool for in vivo cell tracking. It provides the possibility to non-invasively monitor the homing of inflammatory cells towards a site of neural tissue damage. Our aim was to visualize SPIO labeled monocytes entering a brain lesion over time and to compare MR abnormalities with the administration of free label.Cortical lesions were induced in 15 rats by photothrombosis of microvessels. On day 5 after lesion induction, rats received intravenously either no label, free SPIOs or SPIO labeled monocytes. T 2 ⁎ W imaging (4.7 T), sensitive for iron, was performed before, 24 h and 72 h after label injection. In addition, three rats were continuously scanned for 8 h starting directly after administration of free SPIOs.Relative increases of MR abnormalities in rats transplanted with SPIOmonocytes were at 72 h larger compared to controls and free SPIO treated rats. The latter displayed MR abnormalities covering the total lesion area at 24 h. Images from the 8 h scans showed MR abnormalities already 1 h after administration of free SPIOs, indicative for leakage of SPIOs over a damaged blood brain barrier. Our results demonstrate that we are able to visualize the infiltration of monocytes and monitor blood brain barrier permeability, using SPIO labeled monocytes and free SPIOs respectively. These imaging strategies are a valuable tool to study the dynamic pattern of monocyte infiltration in neuroinflammation and may contribute to the development of cell-directed therapeutics. PP06-30 Visualization of effector cell activation within autoimmune CNS lesions after soluble antigen treatment F. Odoardi, K. Rune, C. Cordiglieri. N. Kawakami, H. Wekerle, and A. Flügel Department of Neuroimmunology, Max Planck Institute for Neurobiology, Martinsried, Germany Using real time two-photon imaging we visualize the effects of intravenous soluble antigen on retrovirally labeled MBP-specific CD4 + T cells in acute Experimental Autoimmune Encephalomyelitis (EAE) lesions of the Lewis rat. Recently we found that effector T cells in the diseased CNS show two distinct motility patterns: the majority of the cells (65%) moved fast (maximal speed 25 μm/min) and apparently non directed through the compact tissue (motile cells); a second group of effector T cells (35%) was tethered to a fixed point, forming immune synapse-like structures (stationary cells).We now report that intravenous MBP infusion profoundly changes this T cellular locomotion pattern. The velocity of the effector T cells slowed down dramatically and the percentage of stationary cells increased to >75%. These changes were first observed in the meningeal areas starting 1 h after injection, followed by stopping of T cells within the CNS parenchyma 1 h later. Tethering was paralleled by strong up-regulation of pro-inflammatory cytokines and activation markers of the autoaggressive effector T cells. Furthermore, 3-4 h following the infusion of MBP, clinical EAE became worsened with increased weight loss and paralysis.These results elucidate the speed and efficiency of antigen recognition in vivo and add to our understanding of T cell-mediated autoimmunity and its therapy. PP06-31 A plea for proteomics in biomarker research: Application of protein two-dimensional gel electrophoresis, liquid chromatography and mass spectrometry imaging in rat experimental autoimmune encephalomyelitis Introduction: At Biomed, research is focused on the autoimmune disorder multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). MSI is a newly developed technology that utilizes matrix-assisted laser desorption/ ionisation (MALDI) mass spectrometry to profile and map proteins present in tissue sections. The goal of this study is to identify differentially expressed proteins that can ultimately be used as biomarkers for MS. Methods: Myelin basic protein (MBP)-immunized Lewis rats were sacrificed and brain extracts were subjected to two-dimensional gel electrophoresis (2D-GE) and 2D liquid chromatography (2D-LC), both combined with tandem mass spectrometry. For MSI, cryostat coronal sections of 10 μm were cut, transferred to a stainless steel MALDI target plate and sprayed with matrix. Spectra of 4-25 kDa were acquired using a Bruker Ultraflex II MALDI-TOF/TOF instrument. Results and perspectives: The application of 2D-GE and 2D-LC resulted in the identification of >200 proteins, some of which were found differentially expressed in the brain of EAE animals. Ongoing MSI experiments include the monitoring of selected brain proteins with experimentally determined masses such as myelin basic protein. Future experiments will focus on the identification of differentially distributed proteins in acute EAE as well as in a demyelinating animal model. Differentially expressed proteins found in either proteomic application will be validated for their use as biomarkers in MS. PP06-32 Immunization of mice with the neuronal protein tau induces tauopathy-like pathology and neurological deficits AD-tau aggregation into neurofibrillary-tangles. To investigate this possibility we actively immunized mice with tau protein and tested whether a neurodegenerative disease is induced. Methods: We immunized C57BL mice with recombinant human tau protein with complete Freund's-adjuvant (CFA) and pertussis. Results: Out of 11 female animals immunized with tau 6 developed neurological signs such as limp tail and limb paralysis, whereas none of the CFA-immunized controls showed any symptoms. In all of the tauimmunized mice we have detected anti-tau Abs in serum, and glial activation as well as tau-related pathology in the spinal cord (and to a lesser extent also in brains). These findings were much more prominent in the clinically affected animals. Mononuclear infiltrates and axonal damage were detected only in the clinically affected tau-immunized animals. Conclusion: We demonstrated a neurodegenerative disease induced by immunization with the tau protein. To our best knowledge this is the first report indicating that tau protein has an immunogenic potential, and can induce neuroinflammation and neurodegeneration. These findings may shed light on the possible involvement of tau autoimmunity in neurodegenerative diseases, and point to the potential danger of therapeutic immunization with tau protein. PP06-34 Antigen confirmation contributes to lesion distribution and clinical signs in relapsing remitting MOG-induced EAE Multiple sclerosis (MS) is a chronic disease in which repeated episodes of inflammatory demyelination are associated with an increasing burden of axonal injury, the major cause of chronic disability. To investigate the neuronal response to axonal injury in the context of inflammatory demyelination we have developed a model of relapsing remitting MOGinduced EAE in the Lewis rat characterised by a predictable and focal development of lesions in the cervical spinal cord and cerebellum. Disease is induced by immunisation with refolded recombinant rat MOG in incomplete Freunds adjuvant (IFA) which unlike classical models of disease is characterised by circling and ataxia. Typically 2 or 3 distinct relapses are observed over a period of 70 days post immunisation with lesions appearing to preferentially target the dorsal column of the cervical spinal cord at early time points with subsequent involvement of cerebellar region of the brain. Preliminary studies indicate local differences in the recruitment and activation of a minimal encephalitogenic T cell response that is amplified by the high titre demyelinating antibody response induced by refolded MOG. This model of EAE will be useful for studying regional differences in the response of the blood brain barrier, axons and defined neuronal cell populations to inflammatory demyelination during relapsing remitting disease. PP06-35 Role of MHC class II expression CD4 + T cells in PLP91-110 induced EAE in HLA-DR3 transgenic mice Chella S. David, Ashutosh Mangalam, and Moses Rodriguez Mayo Clinic College of Medicine, Rochester, MN, USA MHC class II molecules play a central role in the control of adaptive immune response through selection of CD4 + T cell repertoire in the thymus and antigen presentation in periphery. Inherited susceptibility to autoimmune disorders such as multiple sclerosis, rheumatoid arthritis and IDDM are associated with particular MHC class II alleles. Advent of HLA transgenic mice has helped us in deciphering role of particular HLA DR and DQ class II molecules in human autoimmune diseases. In mouse, the expression of class II is restricted to professional antigen presenting cells. However in human, class II is also expressed on T cells unlike mouse T cells. We have generated a 'new' humanized HLA class II transgenic mice expressing class II molecules on a subset of CD4 T cells. The expression of class II on CD4 + T cells is inducible and classII + CD4 + T cells can present antigen in the absence of APCs. Further, using experimental autoimmune encephalomyelitis (EAE), a well-established animal model of multiple sclerosis, we tested the functional significance of these classII + CD4 + T cells. DR3.AEo transgenic mice were susceptible to PLP91-110 induced EAE and showed CNS pathology accompanied by widespread inflammation and demyelination seen in human MS patients suggesting a role for class II + CD4 + T cells in the pathogenesis. Dendritic cells (DC) are professional antigen presenting cells that actively initiate adaptive immune responses or control peripheral tolerance to self-antigens. We have demonstrated that in particular signals provided by antigens that target C-type lectin receptors (CLR) function as uptake receptors for glycosylated antigens to maintain homeostasis and induce tolerance. Concomitant DC maturation induced through Toll-like receptors (TLR) triggering are instrumental in inducing auto-immunity.In multiple sclerosis (MS) self-antigens such as myelin/oligodendrocyte glycoprotein (MOG) may induce neuronal demyelination in experimental autoimmune encephalomyelitis (EAE) when combined with adjuvant that activates TLR.To determine whether altered glycosylation of MOG can disrupt this homeostatic control by deranging the CLR/TLR balance, the glycosylation profile of native MOG from human or marmosets was investigated. CLR are indeed differentially expressed in the healthy and diseased human and marmoset MS brain. The suppression by glycosylated MOG on the development of anti-MOG autoimmunity should form the basis for future therapy aimed at tolerance induction in MS.Poster Session 7: EAE-therapy PP07-01 FTY720, sphingosine 1-phosphate receptor modulator, ameliorates experimental autoimmune encephalomyelitis (EAE). Effect of FTY720 on myelin oligodendrocyte glycoprotein-induce EAE in C57BL/6 mice N. Seki a , H. Kataoka a , M. Ohtsuki a , N. Sato a , A. Fukunari b and K. Chiba a a Research Laboratory III, b Discovery Technology Laboratory I, Mitsubishi Pharma Corporation, Yokohama, Japan FTY720 sequesters circulating lymphocytes into secondary lymphoid tissues and thymus by long-term down-regulation of sphingosine 1-phosphate receptor type 1, and is highly effective in myelin proteolipid protein (PLP)induced experimental autoimmune encephalomyelitis (EAE) in SJL/J mice. In this study, we evaluated the therapeutic effect of FTY720 on myelin oligodendrocyte glycoprotein (MOG)-induce EAE in C57BL/6 mice. [Results] EAE was developed 12 days after immunization of MOG to mice. EAE-established mice were used 17 days after MOG immunization. EAEassociated symptoms were maintained during administration period in the control group. Consistent with PLP-induced EAE in SJL/J mice, MOGinduced EAE was significantly inhibited when FTY720 was administered therapeutically at 0.1 to 0.3 mg/kg orally. The infiltration of CD4 + T cells was more markedly than that of CD8 + T cells in the spinal cords in control EAE mice. In FTY720 groups, the area of demyelination and the infiltration of CD4 + T cells and CD8 + T cells in the spinal cords were decreased compared to control. The elevation of IFN-γ mRNA level in the spinal cord was significantly inhibited in FTY720 groups. In addition, since the elevation of IL-17 mRNA was observed in the spinal cords in EAE mice, we are now analyzing the level of IL-17 mRNA in FTY720 groups. [Conclusion] These results suggest that FTY720 shows a therapeutic effect on MOG-induced EAE in C57BL/6 mice by inhibition of Th1 cell infiltration dominantly. PP07-02 FTY720, sphingosine 1-phosphate receptor modulator, ameliorates experimental autoimmune encephalomyelitis (EAE). Effect of FTY720 and FTY720-phosphate on myelin proteolipid protein-induce EAE in SJL/J mice H. Kataoka a , K. Sugahara a , K. Shimano a , Y. Maeda a , M. Koyama b , A. Fukunari b , and K. Chiba a a Research Laboratory III, b Discovery Technology Laboratory I, Mitsubishi Pharma Corporation; Yokohama, Japan Introduction: FTY720, a new class of immunomodulator, is effectively phosphorylated to FTY720-phosphate (FTY720-P), sequesters circulating lymphocytes into secondary lymphoid tissues and thymus by long-term down-regulation of sphingosine 1-phosphate receptor type 1, and exerts immunosuppressive activity in various allograft and autoimmune disease models. In this study, we evaluated the effect of FTY720 on myelin proteolipid protein (PLP)-induce experimental autoimmune encephalomyelitis (EAE) in mice. and clinical score of EAE was assessed periodically. [Results] EAE was developed 11 days after PLP immunization and relapsed 2 weeks after primary EAE in control group. The development of PLP-induced EAE was almost completely prevented and infiltration of CD4 + T cells into spinal cord was decreased by prophylactic treatment with FTY720 at 0.1 to 1 mg/kg orally and FTY720-P at 0.1 to 1 mg/kg intraperitoneally. When FTY720 or FTY720-P was given after establishment of EAE, the relapse of EAE was markedly inhibited as compared with interferon-β, and the area of demyelination and the infiltration of CD4 + T cells were decreased in spinal cords of EAE mice. Conclusion: These results indicate that FTY720 exhibits not only a prophylactic but also a therapeutic effect on PLP-induced EAE in SJL/J mice255and that the effect of FTY720 on EAE appears to be due to a reduction of the infiltration of myelin antigen-specific CD4 + T cells into the inflammation site. PP07-03 Roles of S1P-receptors in the brain: Implications for FTY720 in Multiple Sclerosis Sphingosine-1-phosphate receptors (S1P-Rs), in particular the S1P1-R is expressed on T-cells and is responsible for T-cell egress from lymphatic tissue. Binding of the S1P-mimick FTY720-P to S1P1-Rs on T-cells causes receptor internalization which in turn prevents the trafficking of T-cells to the blood and CNS. This reduces the accumulation of harmful T-cells in the CNS of Multiple Sclerosis patients and phase II studies with FTY720 have clearly shown protective effects in Multiple Sclerosis. Intriguingly, the five S1P-Rs are also differentially expressed on the four major cell types of the brain, namely Neurons, Oligodendrocytes, Astrocytes, and Microglia. Little is known about the role of S1P-Rs in the brain, but studies using S1P and activation of S1P-Rs indicate that these receptors may modulate all four cell types. FTY720 readily crosses the blood-brain barrier. We therefore suggest that FTY720 works beyond the immune system and plays additional protective roles on CNS cells in Multiple Sclerosis. Here, we present data on the effects of FTY720 on the nervous system using in vitro cell culture assays as well as preliminary experiments with direct brain administration of doses that do not cause peripheral lymphocyte depletion at different stages of relapsing remitting experimental autoimmune encephalomyelitis (EAE) in DA rats. PP07-04 Superagonist peptide-induced T cell tolerance for the treatment of multiple sclerosis Kazuyuki Kawamura a , Meike Huebener a , Christopher Self b , Robert Weissert c , and Thomas G. Forsthuber a a University of Texas at San Antonio, San Antonio, USA; b Provid Pharmaceuticals Inc., North Brunswick, NJ, USA; c Hertie Institute for Clinical Brain Research, Tübingen, Germany Specific object: Most of the attention has been focused on T cells specific for high-affinity MHC-binding myelin epitopes in MS patients, such as MBP 85-99, but clinical trials targeting these T cells showed only limited success. A recent study showed that myelin-reactive T cells in MS patients are primarily high-avidity T cells specific for low-affinity MHC-binding epitopes. Studies with B10.PL mice suggested that T cells specific for low-affinity MBP Ac1-9 can be deleted by analogues of this peptide with enhanced binding-affinity for MHC (superagonist). We have therefore investigated whether superagonist peptides can be designed for MBP 115-126 for the treatment of MS. Methods: Superagonist peptide analogues of MBP 115-126 were generated that bound several log-fold better to MHC than the native peptide. Their potential of deleting MBP 115-126-specific T cells was examined in vitro.Results: Some of the designed superagonist peptides were as much as 10 4fold as stimulatory as native MBP 115-126 for MBP 115-126-specific T cells. 70% of the cells stimulated with the superagonist peptides were positively stained for Annexin-V 48 h after activation, indicating induction of apoptosis. Conclusions: Superagonist peptide analogues may be a feasible therapeutic approach to delete encephalitogenic T cells specific for low-affinity HLA-DR-binding peptides. PP07-05 Epicutaneously induced TGF-β-dependent tolerance inhibits experimental autoimmune encephalomyletis Monika Tutaj, Marian Szczepanik Department of Human Developmental Biology, Jagiellonian University, College of Medicine, ul. Kopernika 7, 31-034 Kraków, PolandMultiple sclerosis (MS) is an autoimmune disorder of the central nervous system (CNS) with limited treatment modalities. tolerance induction in the prevention of CNS autoimmunity, we utilized an animal model of multiple sclerosis: experimental autoimmune encephalomyelitis (EAE) induced by immunization with myelin basic protein (MBP). This therapeutic effect was transferable to naïve recipients with lymph node cells from mice treated epicutaneously. These tolerogenic regulatory cells were found to be antigen non-specific, as suppression of EAE also occurred when the antigens such as OVA or TNP were applied e.c. Skin-induced regulatory cells belong to the population of TCRαβ + CD4 + CD8 + lymphocytes. The mechanistic basis for the tolerance was found to be the production of TGF-β by this cells population. These data demonstrate that e.c.-induced regulatory T cells are potent inhibitors of antigen-specific T cell responses, and suggest that e.c. tolerization may have potential effectiveness in the treatment of autoimmune disorders. Recombinant, two domain major histocompatibility complex (MHC) molecules consisting of the peptide binding (α1β1) domains of MHC Class II have potent therapeutic activity in mice with experimental autoimmune encephalomyelitis (EAE). injection, the α1β1 MHCs were only detectable in the serum and plasma of mice for 15-30 min. This short half-life suggested that cellular adherence was responsible for removing α1β1 MHCs from peripheral blood, and indeed, we found that they adhered preferentially to the cell surface of B cells among murine peripheral blood (PB) and spleen (SP) cells. But neither full length (four domain) recombinant MHC Class II molecules nor MHC Class II tetramers had any detectable affinity for PB or SP cells. The FACS detectable level of α1β1 MHC on the surface of B cells decreased after 1 h of incubation; however, Alexa-488 labeled α1β1 MHCs continued to fluoresce from B cells after overnight incubation indicating that the α1β1 MHC was endocytosed. The binding and cellular internalization of α1β1 MHC molecules suggests that the release and acquisition of α1β1 PP07-06 B cells with preferential affinity for partial (α1β1) MHC class II molecules may regulate pathogenic T cells in experimental encephalomyelitis MHCs is a natural and chronic process. This possibility was supported by identifying the transfer of MHC to the surface of B cells in ex vivo cell cultures. We hypothesize that the binding and internalization of α1β1 MHCs by cells may trigger an immunomodulatory response to the peptides bound by the α1β1 MHC. PP07-07 Enhancement of regulatory T cells function via epitope specific immunotherapy leads to disease control in EAE R. Billetta 1 , M. Omori 1 , A. Kaspar 1 , N. Ghahramani 1 , D. Brigham 1 , C. Meschter 2 , P. Lanza 1 , S. Albani 3 Present therapeutic approaches to autoimmune disease largely rely on the direct translation to human therapy of the model trigger antigens used in induction of the animal model.We have identified a mechanism, which modulates inflammation independent of the disease-triggering antigen in autoimmunity based on the recognition by T cells of epitopes derived from heat shock proteins (HSP). Our therapeutic approach aims at restoring T cell mediated regulatory mechanisms, impaired in autoimmunity, by tolerizing to the pro-inflammatory HSP epitopes via mucosal treatment.This study aimed at modulating adaptive Tregs using an HSP-derived peptide via mucosal treatment in monotherapy or combination therapy with Copaxone® after EAE induction in Lewis rats. Our data show that in MBP-immunized rats, EAE development is suppressed clinically by mucosal administration of a particular HSP-derived peptide. We find that the regulatory mechanism activated involves a change in cytokine expression from inflammatory (IFN-gamma and TNF-alpha) to a regulatory profile (IL-4 and IL-10). Additionally, we observed an upregulation of FoxP3 expressing CD4 + CD25 + T cells, indicating a restoration of the regulatory pathway. Histological data were consistent with clinical effect and immunological findings using both mono-and combination therapy.These data suggest the existence of immune regulatory circuits that can be activated by antigens unrelated to the induction of autoimmune disease. The existence of such regulatory pathways suggests that HSP peptidesmediated restoration of Treg function could be of significant therapeutic value for treatment of multiple sclerosis. PP07-08 NK cell-dependent tolerance of experimental autoimmune encephalomyelitis induced by heat shock protein 70-peptide complexes (Hsp70-pc)The hsp70 and hsp70-pc complexes were isolated from brains of health mice or mice with EAE and purified using affinity chromatography with ATP-or ADP-agarose column (respectively).We have show that hsp70-peptide complexes (hsp70-pc) isolated from brains of mice with EAE prevented the development of EAE clinically and pathologically when administered before proteolipid protein 139-151 (PLP 139-151 ) immunization. In animals in which EAE had been suppressed by hsp70-pc, lymphocytes showed increased cell death in response to PLP 139-151 that correlated with elevated IFNγ and NO production. Coculture of spleen cells from hsp70-pc immunized mice with spleen cells from untreated EAE mice, in addition to depletion experiments, showed that NK cells reduced reactivity to PLP 139-151 . Transfer of NK cells from hsp70-pc immunized mice to recipients sensitized for EAE, abolished disease development.In conclusion, we have demonstrated that peptides derived from inflamed CNS and bound to hsp70 are able to induce a novel regulatory circuit involving NK cells inhibiting autoreactive T cells. These findings might further contribute to our understanding of hsp immunoregulatory function in autoimmune diseases. PP07-09 Neonatal neuroantigen-specific Th1 immunity protects from EAE Harald H. Hofstetter 1 , Andra Kovalovsky 1 , Carey L. Shive 1 , Paul V. Lehmann 1 , and Thomas G. Forsthuber 1,2 1 Case Western Reserve University, Department of Pathology, USA; 2 University of Texas at San Antonio, Department of Biology, San Antonio, TX 78249, USA Rationale: The neonatal immune system is believed to be biased towards Th2 immune responses. Consistent with this view, injection of neonatal mice with neuroantigens induces Th2 immunity and protects from induction of experimental autoimmune encephalomyelitis (EAE). In this study we asked whether neuroantigen-specific Th1 immunity could be induced in neonatal mice, and what the outcome would be for autoimmune disease. Methods: Neonatal SJL and B10.PL mice were injected with myelin antigens in CFA and cytokine profiles of the induced T cell responses were measured by ELISPOT assay. Neonatally injected animals were observed for EAE, or the mice were re-injected with myelin antigens as adults to determine protection from EAE. Results: The results show that injection of neonatal SJL and B10.PL mice with PLP139-151 or MBPAc1-11 in CFA resulted in vigorous antigenspecific production of proinflammatory cytokines, including IFN-g and IL-17, but not Th2 cytokines, such as IL-5. Importantly, the neonatally injected mice did not develop EAE, despite the abundant neuroantigen-induced production of proinflammatory cytokines, and were protected from induction of the disease upon reinjection with neuroantigen as adults. Conclusions: The data show that a developmental window exists during the neonatal period in which proinflammatory autoreactive T cells that are not pathogenic can be induced. The data suggest that this could be due to a tissue-specific decrease in the propensity to differentiate towards encephalitogenic "T H -17" cells. The results may provide a rationale for the persistence of autoreactive T cells in healthy individuals in the absence of autoimmune pathology. PP07-10 Targeting FcgammaReceptors in the treatment of EAE Pellkofer H.L. 1 , Schaefer B. 2 , Breithaupt C. 2 , Huber R. 2 , Jacob U. 2 Multiple Sclerosis (MS) is a chronic inflammatory, presumably autoimmune, demyelinating disease of the central nervous system. In particular there is increasing evidence that auto-antibody production by Blymphocytes which result in targeting the myelin sheath by the complement system as well as Fc-receptor carrying cells may play an important role in the immunopathogenesis of demyelination.Our research is focused on the immunemodulatory potential of antagonists and agonists of Fc-receptors and their influence on the onset and disease course of the widely accepted animal model of MS, the experimental autoimmune encephalomyelitis (EAE). In this animal model disease is induced by immunization with the heterologous myelin oligodendrocyte glycoprotein (MOG). Therefore we administered soluble Fc-receptor (FcR), to diminish the interaction of low affinity FcRs with immune complexes. Additionally an antibody was given targeting the inhibitory FcγRIIb and leading to crosslinking with an opposing activatory FcγR which results in inhibition of the immune response by FcR carrying cells.In this study we investigated the potential effects of soluble FcR and anti-FcγRIIb antibody on the disease course of EAE. Treatment with FcR and anti-FcγRIIb after onset of clinical symptoms resulted in a faster recovery and significant reduction of the severity of EAE symptoms. Histopathological examinations revealed that treatment with soluble FcR as well as with the anti-FcγRIIb antibody strongly reduced the amount of infiltrating cells, especially the number of activated macrophages in the spinal cord. Mice receiving soluble FcR also showed decreased levels of pathogenic antibodies and activated B-cells. Our results support the hypothesis that soluble FcR as well as anti-FcγRIIb are an effective therapeutic approach for B-cell mediated autoimmune diseases. PP07-11 Anti-CD20 B-cell depletion reverses EAE induced by MOG protein, but exacerbates disease induced by its encephalitogenic peptide Objective: To investigate anti-CD20 mediated B-cell depletion in experimental autoimmune encephalomyelitis (EAE). Background: B-cells and myelin-specific antibodies (Ab) both play a pathogenic role in CNS autoimmune disease. Recent studies indicate that B cells may also have a regulatory function. We evaluated anti-CD20 mediated B-cell depletion in EAE induced either by MOG p35-55 or by recombinant mouse MOG 1-125 (rMOG). Methods: EAE was induced in human CD20-transgenic C57BL/6 mice in which anti-hCD20-Ab (m2h7) depletes B-cells. In reversal-experiments mice were randomized to treatment at onset of paralysis. T cell proliferation, cytokine and Ab production to MOG p35-55 or rMOG as well as histopathological examination was performed in all mice. Results: Anti-CD20 mediated B-cell depletion had beneficial effects in EAE prevention and reversed paralysis in rMOG-induced EAE. B-cell depleted mice revealed decreased titers for anti-rMOG Ab. In contrast, B-cell depletion prior to immunization exacerbated MOG p35-55-induced EAE with enhanced CNS-infiltration and demyelination and a decreased number of infiltrating B-cells. Splenic MOG 35-55 specific responses were shifted towards a proinflammatory Th1-phenotype with a pronounced decrease in IL-10 production. Conclusion: Anti-CD20 B-cell depletion has differential effects on MOGprotein and MOG-peptide induced autoimmune encephalomyelitis. The beneficial effect of anti-CD20 B-cell depletion in rMOG-induced EAE may reflect a decrease in antigen presentation and/or reduced titers for myelinspecific Ab, whereas exacerbation of MOG peptide-induced EAE may relate to a reduction in B-cell regulation. PP07-12 Neuroprotection and neurogenesis induced by peripheral imunomodulatory treatment of experimental autoimmune encephalomyelitis with glatiramer acetate R. Aharoni, R. Eilam and R. Arnon Immunology Department, The Weizmann Institute of Science, Rehovot, 76100, IsraelIn the autoimmune inflammatory process involved in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), the therapeutic consequences of self neurogenesis and neuroprotection are limited and fail to regenerate functional neurons to compensate the damage. We demonstrate here that the peripheral immunomodulatory treatment for MS/ EAEglatiramer acetate (GA)can enhance neurogenesis and generate neuroprotection in the CNS of EAE inflicted mice.EAE was induced by MOG peptide 35-55 in transgenic mice, which selectively express YFP on their neuronal population and in C57BL/6 mice. GA was injected either concurrently with disease induction or after clinical signs appearance. Neurotrophic factor expression and the in situ manifestations of EAE as well as of GA treatment were analyzed immunohistochemically. Neuroprogenitor proliferation was quantified by the expression of the immature neuronal marker DCX and the incorporation of the in vivo proliferation marker BrdU.In EAE inflicted mice neurotrophins expression and neuroproliferation were elevated following disease appearance, but subsequently declined below that of naive mice. In contrast, GA treatment in various disease stages, led to elevation in BDNF, NT3, NT4 and reduction in the neuronal/axonal damage typical to the neurodegenerative disease course. Moreover, GA augmented the three processes characteristic of neurogenesis, namely neuroprogenitor proliferation, migration and differentiation. The neuroprogenitors manifested massive migration through exciting and dormant migration pathways, into injury sites in brain regions which do not normally undergo neurogenesis. They differentiated to mature neuronal phenotype, endorsing a direct linkage between immunomodulation, neuroprotection, neurogenesis and an in situ therapeutic consequence in the CNS. PP07-13 Transcriptional modulation of the immune response by PPARα agonists ameliorates EAE A.R. Racke ⁎ + Department of Neurology ⁎ and Center for Immunology + , University of Texas Southwestern Medical Center, Dallas, TX, USA Peroxisome Proliferator Activated Receptors (PPARs) are ligandactivated transcription factors belonging to the nuclear hormone receptor superfamily. Rencently, PPARs have been shown to be expressed in macrophages, dendritic cells, T cells, and B cells and play a role in the inflammatory response. Our group has previously shown that the PPARα agonists gemfibrozil and fenofibrate can ameliorate Experimental Autoimmune Encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), by inhibiting IFNγ and promoting IL-4 production. The current study further characterizes the ability of PPARα agonists to induce immune deviation and regulate inflammation in EAE. We found that splenocytes treated with gemfibrozil have altered PPARα, GATA3 and T-bet expression. Using an siRNA specific for PPARα, PPARα and GATA3 levels were reduced. In vivo administration of siRNA-Pparα inhibited the ability of gemfibrozil to ameliorate EAE and prevented IL-4 and IL-5 secretion, suggesting that the agonist is mediating its effects in a receptor-dependent manner. PPARα was found to directly bind the regulatory regions of the IL-4 and IL-5 genes by chromatin immunoprecipitation and IL-4-deficient mice were more suspectible to EAE, even following gemfibrozil treatment. IL-4deficient mice expressed lower levels of PPARα than controls and T-bet, GATA3, and SRC-1 expression were also found to be altered in gemfibroziltreated mice. This data suggests that PPARα agonists mediate their anti-inflammatory effects in an IL-4 and receptor-dependent manner and could potentially be used as a treatment for immune-mediated diseases such as MS. PP07-14 The regulation and production of prostaglandins E2 and D2 in acute and chronic-relapsing rodent models of multiple sclerosis S.S. Ayoub, R. Ashrafi, E.G. Wood, C. Bolton Queen Mary, University of London, London, UK Prostaglandins (PGs) are important pathophysiological mediators involved in a wide variety of processes and, in particular, acute and chronic inflammatory reactions. PG synthesis involves the metabolism of arachidonic acid into short-lived molecules through the actions of the cyclooxygenase (COX) enzymes, COX-1, COX-2 and the recently discovered isoform COX-3. In contrast, PGD 2 has been shown to aid resolution of inflammatory states (Ayoub et al., 2004) .The current study utilised rodent models of experimental autoimmune encephalomyelitis (EAE), the model of the human demyelinating disease multiple sclerosis, to investigate the relationship between PGE 2 and PGD 2 on the initiation and resolution of neuroinflammation. The levels of PGE 2 in central nervous tissues from rodents with EAE were similar to normal values. However, PGE 2 concentrations increased significantly during the recovery phase. PGD 2 content decreased below control values with the development of EAE but returned to normal levels with the resolution of disease.The study will describe the effects of specific receptor antagonists on the expression of COX enzymes and PG production. In particular, the investigation will provide information on the identity of novel pathways involved in the control and production of the PGs in models of EAE.Reference Ayoub, S.S., Botting, R.M., Goorha, S., Colville-Nash, P.R., Willoughby, D. A. and Ballou, L.R., 2004. Acetominophen-induced hypothermia in mice is mediated by a prostaglandin endoperoxide synthase 1 gene-derived protein.Proc. We recently showed that bifunctional compounds comprising nonsteroidal anti-inflammatory (ibuprofen) and cholinergic up-regulation (AChE inhibitor, AChEI; pyridostigmine) moieties ameliorate experimental autoimmune encephalomyelitis (EAE). The presence of cholinergic upregulation moiety increased the anti-inflammatory effect. Consequently, we studied anti-inflammatory effects of acetylcholinesterase inhibitors (AChEI) in CNS inflammation in vivo and in vitro. AChEI suppressed lymphocyte proliferation, pro-inflammatory cytokine production, and extracellular esterase activity. Anti-inflammatory activity was mediated by the alpha-7 nicotinic acetylcholine receptor (neuronal type), the muscarinic receptor had the opposite effect. Treatment of EAE with EN101, an antisense oligodeoxynucleotide, targeted to AChE mRNA, and with conventional AChEI, reduced disease severity and CNS inflammation. Our experimental results suggest that AChEI increases the concentration of extracellular acetylcholine (ACh), rendering it available for interaction with a nicotinic receptor expressed on lymphocytes. These observations point to a novel role for AChEI which may be relevant in CNS inflammatory diseases such as EAE and Multiple Sclerosis. They also emphasize the importance of cholinergic balance in neurological disorders, such as Alzheimer's disease and Myasthenia Gravis, in which these drugs are used. Inhibition of CNS inflammation by cholinergic-up-regulation: A new role for acetylcholinesterase inhibitors Keywords: acetylcholinesterase, acetylcholinesterase inhibitors, α7 nicotinic receptor, CNS inflammation, experimental autoimmune encephalomyelitis, Alzheimer's disease. Multiple sclerosis (MS) is a T cell-mediated auto-immune disease of the CNS that leads to demyelination, axonal damage, and neuronal loss. The pathophysiological mechanisms responsible for development of MS and its animal model EAE are not well defined; however, increased expression/ activation of the calcium-activated neural protease calpain may play a role. Thus, we hypothesize that treatment with a calpain inhibitor will attenuate immune cell infiltration and neurodegeneration in EAE spinal cord. To test the hypothesis, EAE was induced in male Lewis and on days 4 and 8 post-EAE induction, control and EAE rats were treated with the calpain inhibitor SJA6017 (SJA, 3 mg/kg) or vehicle (DMSO). On day 11 post-EAE induction, animals were sacrificed and lumbar spinal cord segments were collected for immunohistolabeling of cell-specific calpain expression (antimcalapin antibody). Calpain was markedly increased in OX42 + (macrophage/microglia) and IFNγ + (T cell) cells in EAE animals, as compared to controls. Since very few cells tested positive for OX42 in control tissue, we suspect that these cells are mainly infiltrating macrophages, with some resident microglia. Treatment with SJA blocked calpain expression and migration of OX-42 + and IFNγ + cells into the CNS. Neuron (NeuN)-specific calpain expression, neuronal death (TUNEL) and axonal damage (de-NFP) were also upregulated in acute EAE spinal cord. These data suggest that calpain inhibition will attenuate immune cell infiltration and neurodegeneration during EAE and may offer potential therapeutic benefits for MS patients. Constitutively expressed HSP90 forms complexes with transcription factor HSF1, and with other client proteins. HSP90 inhibitors promote HSF1 dissociation which induces a heat shock response (HSR), whereas upon dissociation most client proteins are degraded (CPD), including kinase AKT/PKB which is involved in T-cell activation. We showed that clinical symptoms in experimental autoimmune encephalomyelitis (EAE) are reduced by geldanamycin, a naturally occurring HSP90 inhibitor; however whether this involved HSR or CPD was not addressed. We now characterize the effects of novel HSP90 inhibitors on glial and T-cell activation, clinical and histological signs in EAE, and AKT activation. Screening of synthetic HSP90 inhibitors identified several with high (low nM) efficacy to inhibit astrocyte NOS2 and IL1b expression, and T-cell IFNg production. EC72, a geldanamycin derivative, reduced clinical signs of MOG-EAE when given early in disease, and induced recovery when administered to ill mice. In vivo treatment with EC72 did not reduce T-cell activation, nor did EC72 inhibit reactivation of primed T-cells in vitro. However, EC72 and other HSP90 inhibitors reduced CD3:CD28 dependent activation of naïve T-cells, which was also reduced by the AKT inhibitor SH5. HSP90 inhibitors induced AKT degradation, and their effects were lessened in cells expressing constitutively active myrAKT. HSP90 inhibitors could block NOS2 induction in HSF1 null cells. These results demonstrate efficacy of novel HSP90 inhibitors in EAE, and suggest that induction of a HSR is not critical for their effects. Supported in part by a grant from Conforma Therapeutics. Inflammation-induced oxidative stress can lead to axonal degeneration, which is felt to be a major determinant of progressive neurological disability in Multiple Sclerosis (MS). Water-soluble derivatives of fullerenes are a unique class of allotropic form of carbon compounds with potent antioxidant properties. 10 week old mice were immunized with 150 μg of MOG 35-55 peptide in CFA followed by pertussis toxin. Disease is characterized by an attack followed by a progressive phase with chronic clinical impairment. Following the first attack, animals were distributed into different groups with similar disease courses and treated intraperitoneally either with 200 μl of a 1 μM fullerene solution or vehicle (DMSO 2% in PBS) every day until termination of the experiment on day 63. We tested three different fullerene derivatives (ABS-75, ABS-16, and the C60 fullerene core) and found that ABS-75 treatment initiated after disease onset reduced the clinical progression of chronic EAE in NOD mice (treated vs. control, p < 0.05). Fullerene ABS-75 consists of a C60 carbon fullerene core to which a known NMDA receptor ligand was attached. ABS-75 treatment also reduced axonal loss and demyelination (as evaluated by silver and Luxol fast blue staining, respectively) in the white matter of mice. Our data demonstrate a neuroprotective effect of a treatment with a fullerene compound combined with a NMDA receptor ligand that may have applicability in the treatment of progressive MS and other neurodegenerative diseases. PP07-19 Chronic therapeutic administration of natalizumab reduces clinical severity in a guinea pig experimental autoimmune encephalomyelitis model Natalizumab is a humanized monoclonal antibody against alpha-4 integrins for use in multiple sclerosis. Its mechanism of action involves blocking binding of lymphocyte-expressed α4β1 and endothelial VCAM-1, thereby inhibiting extravasation of leukocytes into the central nervous system. Natalizumab is cross-reactive for guinea pig α4β1, and can be dosed long-term (>30 days) in guinea pigs without apparent ill-effect or loss of potency. Groups of 15 early chronic stage EAE-afflicted guinea pigs (15 days post-induction, as chronic progression is beginning) were treated subcutaneously every other day for 56 d with 30 mg/kg natalizumab or vehicle. Natalizumab induced peripheral blood lymphocytosis in EAE animals (mean elevation = 4-fold on d28 and 3.5-fold on d56 relative to vehicle EAE animals). Natalizumab induced an immediate reversal in clinical scores, enhanced body weight gain and reduced mortality relative to vehicle, and maintained significant clinical protection throughout completion of the study. Mean paralytic clinical scores of natalizumab-treated animals on d28 and d56 were 0.86 and 0.4 respectively, compared to vehicle EAE clinical scores of 2.23 and 2.62 for d28 and d56. The percentage of lesioned area in white matter was lower in natalizumab-treated animals than in vehicle EAE animals. These results are consistent with human clinical data demonstrating reduced clinical severity (relapse rate and disability score) and reduction in gadolinium-enhancing lesions by MRI. Further characterization of lesions in the guinea pig model will be investigated using cell-type-specific antibodies to identify infiltrating cell types via immunohistochemistry. PP07-20 Fc-receptor antagonists are effective in the therapy of autoimmune disease animal models Fc-receptors of IgG (FcγR) play a crucial role in antibody-mediated autoimmune diseases by linking the humoral to the cellular immune response. Therefore we investigated in animal models whether the administration of soluble Fc-receptors (sFcR) as antagonists of immune complex recognition could influence the course of autoantibody mediated autoimmune disorders.In vitro investigations show that sFcR bind to human and mice pooled IgG, inhibit antibody mediated phagocytosis of red blood cells and are strong antagonists of immune complex binding to B-cells.Finally, in murine models of Multiple Sclerosis (MS), Idiopathic Thrombocytopenic Purpura, Rheumatoid Arthritis and Systemic Lupus Erythematodes treatment with sFcR significantly reduces the severity of symptoms thereby demonstrating the model independency of the approach. Histopathological examinations of the spinal cord of sFcR treated mice in experimental autoimmune encephalomyelitis revealed a decreased amount of infiltrated cells and especially reduced numbers of activated macrophages. Furthermore treatment resulted in decreased demyelinated plaques and axonal damage. Mice receiving the soluble receptor also showed lower levels of pathogenic antibodies and less activated B-cells. Multiple Sclerosis (MS) is a major chronic autoimmune pathology of the central nervous system. Members of the matrix metalloproteinase (MMPs) family are known to be important factors involved at key steps of MS, however little is known of the role of MMP12 in MS. To determine the extent to which MMP12 might be involved in this pathology, we administered orally a MMP12 inhibitor in preventive regimen (3, 10, 30 mg/kg) in the MOG-induced chronic mice as well as in curative regimen (1.5, 5, 15, 30 mg/kg) in the whole spinal cord protractedremitting rat experimental autoimmune encephalomyelitis (EAE) models which are animal models mimicking various aspects of MS. In preventive treatment, the MMP12 inhibitor significantly delayed the disease onset and decreased the severity of the clinical signs over the course of the pathology suggesting the involvement of MMP12 in the induction as well as the following effector phase of the pathology. In the protracted-remitting EAE model, a mixed model of inflammation and early demyelination, oral administration starting at clinical score 1 decreased the severity of the first attack and the following remitting phases at a minimal dose of 5 mg/kg. The aim of this study was to examine the effect of TLR4 stimulation in EAE, an animal model of multiple sclerosis (MS). Upon recognition of bacterial LPS, TLR4 signaling induces the expression of a range of proand anti-inflammatory mediators. The role of TLR4 in autoimmunity is not well characterized. We induced chronic EAE in female C57BL/6 mice by subcutaneous immunization with myelin oligodendrocyte glycoprotein (MOG35-55) in CFA and intraperitoneal (i.p.) administration of pertussis toxin on days 0 and 2. on days 0-5 and were sacrificed at various timepoints. Spleen cells were cultured with or without antigen and assessed for proliferation. Culture supernatants were assayed for cytokine levels by cytometric bead arrays and ELISA. Spinal cord cellular infiltration was characterized by flow cytometry. Regardless of antigenic stimulus in vitro, the monocyte/macrophage chemoattractant, MCP-1 (CCL2), was elevated in splenocyte culture supernatants of LPS treated mice compared to PBS controls at day 19 post immunization (p.i.). Despite significantly greater T cell proliferation in LPS treated mice at day 6 p.i., there was lower inflammatory infiltration of the spinal cord of these mice at day 19 p.i. We conclude that stimulation of TLR4 delays the onset of chronic EAE by mechanisms which include inhibition of lympho-mononuclear cell infiltration into the CNS. This may be due to enhanced chemotactic activity of CCL-2 in the peripheral immune system. PP07-23 Argatroban, a direct thrombin inhibitor, suppresses the severity of experimental autoimmune encephalomyelitis Experimental autoimmune encephalomyelitis (EAE) is an inflammatory and demyelinating autoimmune disease of the central nervous system (CNS) that provides a model for human multiple sclerosis (MS). Numerous studies have proved the correlation between the coagulation-fibrinolysis system and the pathogenesis of EAE. We have examined the role of coagulation system in EAE by using argatroban, which is a direct inhibitor of thrombin. Plasma thrombinantithrombin Ø complex (TAT) values were significantly lower in argatroban treated rats compared with saline-treated controls (p < 0.01). Immunohistologically, perivascular fibrin depositions were reduced in lumbosacrococcygeal spinal cord of guinea-pig myelin basic protein (GPMBP)-sensitized rats treated with argatroban. RT-PCR revealed that the cytokine levels was reduced within spinal cord of argatroban treated rats compared with control rats, showing cell migration is suppressed by inhibition of fibrin deposition. Furthermore, our data suggest that fibrin could be a potential therapeutic target in MS. Anticoagulation therapies aiming selectively inhibit the fibrin deposition might be a safer approach than the exogenous administration derived from organisms. PP07-24 Vitamin K2 ameliorates experimental autoimmune encephalomyelitis in Lewis rats M. Moriya, Y. Nakatsuji, T. Okuno, S. Sakoda Department of Neurology, Osaka University Graduate School of Medicine, Suita, Japan Vitamin K 2 (VK2) has been widely used for the management of osteoporosis in Japan. It was demonstrated that HMG-coA reductase inhibitors (statins) have beneficial effects on experimental autoimmune encephalomyelitis (EAE), and the inhibition of geranylgeranyl-pyrophosphate (GGPP) synthesis plays a key role in the effects. Since the side chain of VK2 has a common structure to geranylgeranyl-pyrophosphate (GGPP), it could be expected that VK2 has similar effects as seen in statins. Lewis rats were actively immunized with myelin basic protein (MBP). Each rats received intraperitoneal administration of VK2 and clinical scores were evaluated daily. Histological, immunohistochemical, and Western blot analyses of the spinal cords and cultured glial cells were also performed. The clinical severity of EAE was significantly ameliorated by the prophylactic administration of VK2. Histological analysis revealed that inflammatory cellular infiltration was significantly reduced in the spinal cords of VK2treated rats. Immunohistochemical and Western blot analyses revealed that the expression of both MHC class II and inducible nitric oxide synthase (iNOS) were significantly reduced in the spinal cords of VK2-treated rats with EAE. The inhibitory effect of VK2 on the iNOS expression in glial cells was also observed in vitro. Considering the wide use of VK2 without noticeable untoward effects, it may be applicable to the patients with MS. Background: The TNF-related weak inducer of apoptosis (TWEAK) mediates proinflammatory effects by its receptor fibroblast-growth-factorinducible-14 (Fn14). We previously showed that TWEAK/Fn14-interactions contribute to lesion development in experimental autoimmune encephalomyelitis (EAE) and that neutralisation of TWEAK or Fn14 ameliorates CNS inflammation in EAE. TWEAK also increased CCL2 release by CNS resident cells.Objective: Revealing the mechanism(s) of TWEAK/Fn14 promoting autoimmune inflammation.Methods: Neutralising TWEAK/Fn14-antibodies were induced by vaccination with the extracellular domain of TWEAK or full length Fn14. Adoptive transfer EAE (AT-EAE) was induced in SJL mice by transfer of PLP-specific lymph node cells (LNCs). C57Bl/6 CCL2 +/+ or CCL2 −/− mice were immunised with MOG-peptide to induce active EAE. Chemokine release in culture supernatants was measured by ELISA.Results: AT-EAE using vaccination of either donor or recipient mice showed that the primary immune response and T cell encephalitogenicity are not modulated by TWEAK/Fn14. However, Fn14-vaccination of Tcell-recipient mice resulted in significant amelioration of EAE-signs suggesting the blood-brain-barrier as a potential site of action. Vaccination against TWEAK or Fn14 protected both CCL2 +/+ andto a lesser extent -CCL2 −/− mice from the development of EAE indicating that the effect of TWEAK/Fn14-blockade on lesion development is only partially dependent on CCL2. Finally, data will be presented showing the influence of TWEAK/Fn14 on migration of mononuclear cells in an in vitro transwell system.Conclusion: TWEAK/Fn14 might promote lesion development via several mechanisms. While TWEAK-induced modulation of CCL2 and other proinflammatory molecules by TWEAK may be involved, TWEAK does not alter the encephalitogenic T cell response. PP07-26 Nucleoside analogues effect on glial response in experimental autoimmune encephalomyelitis We have previously shown that combined treatment with nucleoside analogues (ribavirin -R + tiazofurin -T), inosine monophosphate dehydrogenase inhibitors, ameliorates clinical signs and histological lesions of EAE in susceptible rats, when they are given preventatively. The aim of this study was to investigate the effect of combined treatment with R + T, given with the appearance of first EAE clinical sign, on microglia and astrocytes response. These cells of the target tissue also participate in an autoimmune process. The disease was induced in Dark Agouti rats with rat spinal cord homogenate and had acute monophasic course. Ribavirin and tiazofurin were given at a dosage of 30 mg/kg/day and 10 mg/kg every other day, for 15 days, respectively. Amelioration of clinical signs and faster recovery was shown in group treated with combination of R and T in comparison to control group. Immunohistochemical analysis of the spinal cord tissue isolated after 15 days of combined therapy revealed decrease in vimentin positive cells and microglia compared to control group. Additionally, morphology of GFAP positive (glial fibrillary acid protein) cells and microglia indicated to reactive type of these cells in control group. Results of this study revealed that R and T modulate glial response and have EAE protective effects when they are given from the onset of disease. (This work was supported from Ministry of Science and Environmental Protection, Republic of Serbia, Serbia and Montenegro, Grants 143005, 143029 and 145066) PP07-27 Multiple neuroprotective mechanisms of minocycline in autoimmune CNS inflammation Katharina Maier a , Doron Merkler b , Joachim Gerber a , Naimeh Taheri a , Antje V. Kuhnert a , Sarah K. Williams a , Clemens Neusch a , Mathias Bähr a , and Ricarda Diem a a Neurologische Universitätsklinik; b Institut für Neuropathologie, Göttingen, GermanyAxonal destruction and neuronal loss occur early during multiple sclerosis, an autoimmune inflammatory CNS disease that frequently manifests with acute optic neuritis. Available therapies mainly target the inflammatory component of the disease but fail to prevent neurodegeneration. To investigate the effect of minocycline on the survival of retinal ganglion cells (RGCs), the neurons that form the axons of the optic nerve, we used a rat model of myelin oligodendrocyte glycoprotein (MOG)induced experimental autoimmune encephalomyelitis. Functional and histopathological data of RGCs and optic nerves revealed neuronal and axonal protection when minocycline treatment was started on the day of immunization. Furthermore, we demonstrate that minocycline-induced neuroprotection is related to a direct antagonism of multiple mechanisms leading to neuronal cell death such as the induction of anti-apoptotic intracellular signalling pathways and a decrease in glutamate excitotoxicity. From these observations, we conclude that minocycline exerts neuroprotective effects independent of its anti-inflammatory properties. This hypothesis was confirmed in a non-inflammatory disease model leading to degeneration of RGCs, the surgical transection of the optic nerve. PP07-28 Combining immunotherapy with strategies to promote myelin repair for the treatment of established EAE The goals of the current project are to investigate the cellular and molecular mechanisms regulating the processes of demyelination and remyelination during CNS autoimmune disease and to design immunoregulatory and glial regenerative combinatorial therapies for the treatment of established disease in animal models of MS. Real-time PCR data suggest a correlation between myelin gene expression in the CNS and the distinct phases of relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE). Compared to preclinical stages, we have observed decreased myelin gene expression during acute disease and first relapse and increased Notch signaling molecule expression during R-EAE. Notch inhibits oligodendrocyte differentiation and myelination during development and has been hypothesized as a limiting factor for remyelination. We have designed a treatment strategy for ongoing EAE that combines γ-secretase inhibition (which blocks Notch signaling in addition to a host of other factors) and anti-CD80(Fab), a wellestablished immunoregulatory strategy. We are additionally designing and testing other myelin repair strategies in combination with immunoregulatory techniques. Preliminary analyses of T cell responses and the extent of lesion repair following combinatorial treatments have yielded promising results for the future of MS therapy. PP07-29 Effect of anti-CD81 antibodies in experimental autoimmune encephalitis Infiltration of leukocytes into the CNS is a key event in the formation of new lesions in multiple sclerosis (MS). Molecules involved in the interaction between leukocytes and endothelial cells represent prime candidate targets for novel therapeutic approaches. We have previously shown that the tetraspanin CD81, a regulator of the VLA-4 integrin, is involved in the migration of monocytes across the blood brain barrier in vitro. Here we asked whether inhibition of monocyte migration in vivo by use of anti-CD81 antibodies would have a beneficial effect on neurological symptoms in experimental autoimmune encephalitis. Mice were immunized with PLP139-151 and treated with two different hamster anti-mouse CD81 antibodies, isotype control mAb or vehicle (n = 10 per group). Antibodies were injected i.p. at 100 μg per injection every other day for 3 weeks starting 5 days post immunisation. The results showed that clinical symptoms were reduced by 47% in mice treated with Eat2 mAb as compared to vehicle (cumulative clinical scores 25± 7 vs. 47 ± 9; p < 0.05). Interestingly, treatment with the other anti-CD81 mAb 2F7 had no effect on clinical scores, whereas an irrelevant mAb resulted in intermediate scores. We are now validating these results in additional EAE models and studying the pathogenetic mechanisms underlying the treatment effect. PP07-31 Allogeneic versus syngeneic BMT for prevention of EAE: Role of donor chimerism, donor susceptibility to EAE and alloreactivity Background: Autologous bone marrow transplantation (autoBMT) is explored as a novel treatment for MS. Case reports exist on beneficial effects of allogeneic BMT (alloBMT). However, unresolved questions about the action mechanism warrant investigation in animal models. Aim: To study EAE incidence and disease course after murine BMT and explore the role of donor-EAE-susceptibility, degree of donor-chimerism, and posttransplant alloreactivity. Methods: Allogeneic and syngeneic radiation BM chimeras were prepared using Balb/c (EAE-resistant), C57BL/6 and B6SJLF1 (EAE-susceptible) mice. Some alloBMT groups were given donor-type leukocyte infusions (DLI) to increase donor-chimerism. In all, MOG 35-55 -EAE was induced on day 28 or 42, followed 14 days later by ex vivo MOG 35-55 splenocyte proliferation assays.Results: In C57BL/6 syngeneic chimeras and Balb/c → C57BL/6 low-grade allogeneic chimeras, day-28-induced EAE was prevented in 80% resp. Full donor chimerism (through DLI) in Balb/c → C57BL/6 allogeneic chimeras resulted in complete prevention of day-42-induced EAE, and loss of the MOG 35-55proliferative response.In C57BL/6 → B6SJLF1 high-grade allogeneic chimeras, induced after irradiation, neither prevention of EAE nor loss of MOG 35-55 -specific proliferation were seen. In contrast, non-radiation high-grade C57BL/ 6 → B6SJLF1 chimeras obtained by repetitive DLI (strong in vivo alloreactivity), again resulted in complete prevention of EAE and loss of MOG 35-55 -specific proliferation. Conclusion: The transient EAE-protective effect of synBMT probably relates to posttransplant aspecific immunosuppression. High-grade donorchimerism from an EAE-non-susceptible donor confers robust EAEresistance, indicating the role of genetic susceptibility. However, equally strong protection was obtained using an EAE-susceptible donor, when a non-irradiation model of DLI-induced chimerism was used, suggesting a role for alloreactivity. PP07-32 Suppression of experimental autoimmune encephalomyelitis by transfer of lymphokine-activated natural killer (NK) cells Shigemi Nagayama, Sachiko Miyake, and Takashi Yamamura Department of Immunology, National Institute of Neuroscience, NCNP, JapanObjective: The purpose of this experiment is to explore if passive transfer of natural killer (NK) cells may suppress the development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). NK cells were isolated from the spleen of non-immunized B6 mice, activated in vitro with IL-2, and were inoculated intravenously into MOG-sensitized mice on the indicated days. Results: At first, we studied whether NK cells are infiltrated within lesions of the central nervous system (CNS) of EAE mice. Immunofluorescence analysis of mononuclear cells isolated from the CNS of MOG-sensitized mice on Days 7, 14 or 21 after sensitization demonstrated the presence of NK cells (CD3-NK1.1 + cells), with the positive correlation of the number of migrated NK cells with the clinical severity of EAE. The intravenous injection of in vitro IL-2-activated NK cells (NK-LAK cells) into MOGsensitized mice on Days 7, 14 and 21 after sensitization clearly suppressed clinical severity of EAE. In order to clarify whether passively transferred NK-LAK cells could infiltrate into the CNS lesion, we transferred NK-LAK cells of green mice into EAE-positive B6 mice. We found that NK-LAK cells of green mice could migrate to the CNS of mice developing EAE. Conclusion: These findings showed that transfer of NK-LAK cells could down-regulate the ongoing inflammation in the CNS lesions of EAE. PP07-33 Expansion of CD4 + CD25 + Foxp3 + regulatory T cells in Midkine-deficiency mice results in partial resistance to experimental autoimmune encephalomyelitis: A novel immunomodulation by Midkine Jinyan Wang a , Hideyuki Takeuchi a , Shijie Jin a , Jun Kawanokuchi a , Reiko Kuno a , Yoshifumi Sonobe a , Izumi Yawata a , Yukiko Doi a , Jianfeng Liang a , Guiqin Zhang a , Tetsuya Mizuno a , Hisako Muramatsu b , Takashi Muramatsu b , and Akio Suzumura a a Department of Neuroimmunology, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan; b Department of Biochemistry, Graduate School of Medicine, Nagoya University, Nagoya, Japan Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), is a Th1-mediated inflammatory demyelinating disease of the CNS. CD4 + CD25 + Foxp3 + regulatory T cells (Treg) play crucial roles in the induction of peripheral tolerance by controlling autoreactive T cells. Increasing evidences have shown that Treg cells play key roles on negative regulations the pathogenesis of MS. However, the endogenous factors and mechanisms controlling the peripheral generation of Treg cells are largely unknown.Midkine (MK) is, a 13-KD protein, a member of the heparin-binding growth factors and reportedly exerts pleiotropic activities on cell proliferation, migration, angiogenesis and fibrinolysis in a variety of tissues. This resistance was closely associated with substantial expansion of Treg cells in peripheral lymph nodes after MOG immunization as compared with those in wild mice. Administration of MK to MK deficient mice significantly abrogated the resistance of EAE and suppressed the expansion of Treg cells in vivo and in vitro. In addition, the frequency of autoreactive Th1 cells decreased without affecting Th2 population. These results indicate that MK deficiency results in expansion of Treg cells in periphery and control the activation of autoreactive Th1. Target for MK deficiency may be an effective therapeutic strategy for autoimmune diseases, including MS. In previous studies we have shown that RPE (retinal pigment epithelium) were able to express MHC class II if they were exposed to inflammatory cytokines such as IFN-γ. In this study, we determined the in vitro activation of IRBP (Interphotoreceptor Binding Protein) specific CD4 and CD8 T cells in the presence or absence of RPE cells and the factors produced by nonactivated and activated RPE. We also compared the effect of astrocytes, another eye immuno-competent cell, on the in vitro activation of uveitogenic CD4 and CD8 cells. RPE cells differentially regulate the activation of uveitogenic CD4 and CD8 T cells Our results have showed RPE cells inhibited the high-degree-activated, both IRBP-specific CD4 and CD8 T cells, but enhanced low-degreeactivated T cells; differentially, astrocytes inhibited IRBP-CD4 at both highand low-activated degree, but enhanced low-degree-activated CD8 cells. Moreover, factors produced by activated RPE enhanced IRBP-CD4 but inhibited CD8 T cell activation and the factors produced by activated astrocytes inhibited both T cells. Our results suggest that (1) both RPE and astrocytes have regulatory effects on activation of T cells in the eye. At least, a part of the regulatory effect is mediated by the factors they produce, (2) : different parenchymal cells of the eye may have distinct regulatory effects on CD4 and CD8 autoreactive T cell. Therefore, studies on the mechanism by which parenchymal cells of the eye regulate the invading autoreactive T cells should provide needed information for therapeutic intervention. Estrogen (E2)-induced immunomodulation involves dual effects on antigen presenting cells (APC) and CD4 + CD25 + regulatory T cells (Treg), but not a direct effect on effector T cells. In this report, we further investigated the effects of E2 on APC and Treg cell function. We found that E2 treatment in vivo strongly reduced recovery of APC from the peritoneal cavity and inhibited their ability to induce inflammatory cytokines, IL-12 and IFN-γ, but enhanced secretion of IL-10. Moreover, E2 conditioned bone marrow-derived dendritic cells (BM-DC) could both enhance Treg cell activity and directly inhibit responder T cells in the absence of Treg cells. We examined whether this E2-induced inhibitory activity of BM-DC might involve co-stimulation through the recently described PD-1 pathway. Both E2 and pregnancy markedly enhanced PD-1 expression in several types of APC, including macrophages, B cells, and especially dendritic cells (DC). Similar to E2-induced enhancement of FoxP3 expression and EAE protection, E2-induced enhancement of PD-1 + cells was also mediated through estrogen receptor alpha (Esr1) in DC and macrophages, but not B cells. Based on antibody inhibition studies, PD-1 interaction with its ligands, PDL-1 and especially PDL-2, could mediate either positive or negative regulatory signaling in both mature and immature E2-conditioned DC, depending respectively on a relatively high (10:1) or low (1:1) ratio of T cells:BM-DC. PP08-02 Expression and functional activity of mu-opioid receptors (MOR) on a myelo-monocytic HL-60 cell line J. Gabrilovac, D. Breljak, B. Čupić Ruđer Bošković Institute, Division of Molecular Medicine, Zagreb, Croatia Outside nervous system, opioid receptors were also found on cells of hematopoietic origin in which they participate in modulation of growth, differentiation and functional activity. Based on ligand selectivity there are three main classes of opioid receptors: delta, mu and kappa. Mu-opioid receptors (MOR) are the most important class involved in analgesia mediated by alkaloid opiate morphine. By using polyclonal antibodies to mu-opioid receptors and FACS analysis we have recently reported membrane expression of MOR on a sub-population of HL-60 cells (1) . The functionality of MOR expressed on HL-60 cells was examined by measuring Ca + mobilisation triggered by selective MOR agonists. Morphine and endomorphin-2 were used as representative agonists with alkaloid or peptide structure, respectively. Presence of mRNA for MOR was detected in HL-60 cells, which corresponded to that in a neuronal cell line SH-SY5Y. Both morphine and endomorphin-2 triggered Ca + mobilisation in HL-60 cells in a concentration-dependent manner. Endomorphin-2-induced Ca + mobilisation was reduced by selective MOR antagonist betafunaltrexamine suggesting a MOR-mediated action. In short, cells of immature myelo-monocytic HL-60 line express MOR both at the level of mRNA and at the level of a functionally active protein capable of transmitting intracellular signals via Ca + upon ligation with selective agonists of alkaloid or peptide structure. (1) Gabrilovac J et al., Membrane expression of delta, mu and kappa opioid receptors on HL-60, THP-1 and U937 cell lines. PP08-03 Crosstalk between chemokine, opioid, and vanilloid receptors Chemokine receptors serve as a bridge between the immune and neural systems. Neuropeptides, such as opioids, inhibit chemokine receptor function on leukocytes by activating Gi protein and calcium-independent protein kinase C. Conversely, during inflammation, chemokines similarly regulate neuronal sensing in two ways. Activation of chemokine receptors on neurons desensitizes the analgesic μ-opioid receptor and concomitantly enhances the sensitivity of a pain receptor, TRPV1, via protein kinase Cdependent phosphorylation, resulting in hyperalgesic effects. We will present the cellular, biochemical, and in vivo evidence to show that receptor cross-talk provides an important pathway for neuroimmune interactions. PP08-04 The molecular structure basis and receptor mechanisms of interferon-alpha on hypothalamic-pituitary-adrenal axis Hypothalamic-pituitary-adrenal (HPA) axis plays a pivotal role in responding to immunological challenges. Cytokines can modulate the function of the HPA axis. Interferon-alpha (IFNα) also has this effect, but the mechanisms are not thoroughly characterized, and the results remain controversial and conflict. This study is to explore the molecular structure basis and receptor mechanisms of IFNα upon the HPA axis in vivo and vitro. Two kinds of IFNα mutants were obtained and used, which are 129Ser-IFNα with analgesic activity and 38Leu-IFNα with antiviral activity. After administration of IFNα and its mutants to ventricle in vivo and to cultured hypothalamic slices, AtT20 cells, and adrenal cortical slices, the contents of CRH, ACTH, and corticosterone in samples were assayed respectively. The results showed that Wild-IFNα and 129Ser-IFNα could decrease CRH, ACTH and corticosterone contents in vivo, and decrease CRH content, stimulate corticosterone secretion in vitro significantly, which could be blocked by opioid receptor antagonist naloxone pretreated. 129S-IFNα had no direct effects on ACTH secretion in AtT20 cells without μ opioid receptor expression. It is the first highly suggested that IFNα maybe regulate the HPA axis at least by two different receptor mechanisms: one is opioid receptors and the molecular structure basis is opioid domain of IFNα. PP08-05 Roles of brain IFN-α and 5-HT transporter in immunologically induced fatigue as a model for chronic fatigue syndrome T. Katafuchi Department of Integrative Physiology, Graduate School of Medical Science, Kyushu University, Fukuoka, JapanTo investigate brain mechanisms of fatigue, the immunologically induced fatigue was conducted in rats by intraperitoneal (IP) injection of a synthetic double-stranded RNAs, polyriboinosinic: polyribocytidylic acid (poly I:C). The amount of mRNA for IFN-α measured by real-time capillary PCR method increased in the hypothalamic nuclei and cortex on day 1 and even day 7. Expression of 5-HT transporter, which is reported be induced by IFNα, also enhanced in the same brain regions where IFN-α increased on day 7. In vivo brain microdialysis revealed a decrease in extracellular concentration of 5-HT in the prefrontal cortex after poly I:C, which was blocked by a selective 5-HT reuptake inhibitor, fluoxetine. Finally, the poly I:C-induced suppression of the running wheel activity was significantly attenuated by a 5-HT 1A receptor agonist, but not by 5-HT 2 , 5-HT 3 , or dopamine D 3 receptor agonists. These findings, taken together, suggest that brain IFN-α and 5-HT 1A receptors play an important role in the neuronal mechanisms of the poly I:C-induced fatigue.Reference: Katafuchi T, et al. PP08-07 Induction of microglial apoptosis by corticotropin-releasing hormone: A potential regulatory mechanism of neuroinflammation K. Suk, J. Ock, S. Kim, and J. Lee Department of Pharmacology, Kyungpook National University School of Medicine, Daegu, ksuk@knu.ac.kr Neuropeptides are short-chain peptides found in brain tissue, with some functioning as neurotransmitters and others functioning as hormones. Neuropeptides may directly or indirectly modulate glial functions in the central nervous system (CNS). While vasoactive intestinal peptide, substance P, cholecystokinin, or neuropeptide Y did not affect microglial cell viability, corticotropin-releasing hormone (CRH) induced a classical apoptosis of mouse microglia in culture as evidenced by nuclear condensation and fragmentation, TUNEL staining, and cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP) protein. CRH, however, did not influence nitric oxide production (assessed by Griess reaction) or inflammatory gene expression (assessed by RT-PCR and Western blot analysis) including cytokines and chemokines, indicating that CRH did not affect the inflammatory activation of microglia. Taken together, our results indicate that the stress neuropeptide CRH may regulate neuroinflammation by inducing the apoptosis of microglia, the major cellular source of inflammatory mediators in CNS. Department of Pharmacology -University of Milan, Italy The antiproliferative effect of a somatostatin analog SOM 230 and its capacity to regulate cytokine release were evaluated on human peripheral blood lymphocytes (hPBL) activated by phytohemoagglutinin (PHA), pokeweed (PWM) or alloantigens (MLR). PBL proliferation, measured by thymidine uptake, resulted significantly inhibited aside from the stimulus, but alloantigen stimulation were the most affected by the presence of SOM 230 and while mitogen induced proliferation were inhibited by 10 − 11 M the alloantigen induced one by 10 − 13 M. The profile of cytokine release were evaluated in MLR supernatants by an ELISA test starting at 24 h till five days of culture. To evaluate the growth inhibition of lymphocytes, we studied the DNA distribution in hPBL stimulated by PHA or PWM in the presence of SOM-230 10 − 11 M by propidium iodure incorporation by flow cytometry analysis. After 48 h of stimulation with PHA or PWM in the presence of SOM 230 the cell cycle analysis demonstrated a modest accumulation of cells in the S-phase. This phenomenon were then analysed by 5-bromo-2′-deoxyuridine (BrdU) incorporation for 30 h with no significative results. We are now studying the cell cycle by a longer pulse of BrdU and, on the other hand, we are evaluating if the antiproliferative effect of SOM 230 is dependent either on a apoptotic (by annexin V test) or autophagic effect (by LC3 immunofluorescence test). PP08-09 Regulation of immune cell function by a non-neuronal cholinergic system Y. Moriwaki, K. Yoshikawa, Y. Fujii, and K. Kawashima Department of Pharmacology, Kyoritsu College of Pharmacy, Tokyo, Japan Lymphocytes express all the components required to constitute an independent cholinergic system, such as acetylcholine (ACh), choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and muscarinic and nicotinic ACh receptors (mAChR and nAChR, respectively). Stimulation of mAChRs on T and B cells causes oscillating intracellular Ca 2+ signaling and up-regulates c-fos gene expression. Stimulation of nAChRs produces rapid and transient intracellular Ca 2+ signaling. We investigated the gene expression of cholinergic components in splenic mononuclear cells (MNLs), bone marrow-derived dendritic cells (BMDCs), and macrophages of C57BL/6J mice. In resting condition, ChAT gene expression was detected only in the brain by RT-PCR, while AChE gene expression was observed in all samples. After ConA or LPS stimulation, ChAT gene expression was detected in MNLs and BMDCs, respectively, suggesting that immunological stimulation induces ACh synthesis. Genes for M 1 -M 5 mAChRs were expressed in all samples. Genes for the α2, α5, α6, α7, α10, and β2 subunits of the nAChR were expressed in all samples, while no expression was detected for the genes for α3 and β3. The gene for SLURP-1, a recently discovered endogenous peptide allosteric ligand of α7 nAChR, was expressed in all samples. These results suggest that ACh and SLURP-1 play intermediary roles in the dialogue among lymphocytes, BMDCs, and macrophages during immune responses via mAChR-and nAChRmediated pathways in autocrine and/or paracrine fashion, via contact with each other through cell surface molecules such as ICAM-1/LFA and CD4/MHC-II. Evidence suggests that inflammation is a significant contributor to pathology in a number of neurodegenerative disease states. In this regard, the pro-inflammatory cytokine interleukin-1β (IL-1β) plays a key role in initiating an immune response within the central nervous system (CNS). The actions of IL-1β can be regulated in vivo by interleukin-1 receptor antagonist (IL-1ra); a molecule that prevents IL-1β from acting on the IL-1type I receptor. Consequently, the balance between IL-1ra/IL-1 β is of pathological importance, and pharmacological strategies that tip the balance in favour of IL-1ra may be of therapeutic benefit. Evidence is emerging to suggest that the neurotransmitter noradrenaline elicits anti-inflammatory actions in the CNS, and consequently may play an endogenous neuroprotective role. In this study we report that noradrenaline induces production of secreted IL-1ra from primary rat mixed glial cells, without altering IL-1β concentrations. Noradrenaline-induced IL-1ra production was attenuated by the non-selective β-adrenoceptor antagonist propranolol, and also by the selective β 2 -adrenoceptor antagonist butoxamine, but not by the selective β 1 -adrenoceptor antagonist metoprolol. Furthermore, the selective β 2 -adrenoceptor agonist salbutamol mimicked the ability of noradrenaline to increase IL-1ra production. Taken together these data indicate that the β 2 -adrenoceptor mediates the ability of noradrenaline to increase IL-1ra production. Consistent with the ability of β-adrenoceptors to activate the cAMP-protein kinase A pathway, the stable cAMP analog dibutryl cAMP mimicked, and the PKA antagonist RP-cAMPs blocked the ability of noradrenaline to increase IL-1ra. Overall, these data further demonstrate that noradrenaline is a neurotransmitter with anti-inflammatory and therefore potential neuroprotective actions within the CNS.The authors acknowledge grant support from Science Foundation Ireland. PP08-11 Nicotine controls T cell function through direct interaction and through the CNS Lymphoid tissues are innervated by the sympathetic autonomic nerve terminals, and the role of this innervation in the immune tissues is not clear. Chronic exposure to nicotine causes immunosuppression and impairs T cell function. When given intracerebroventricularly (ICV), relatively small concentrations of nicotine affect T cell responses, and these effects are not significantly influenced by adrenalectomy, indicating that the HPA axis does not play a major role in the nicotine-induced immunosuppression. However, the immunosuppression is blocked by ganglionic blockers, indicating that under these conditions the effects of nicotine are regulated by the sympathetic autonomic nervous system (sANS). Peripheral administration of nicotine at concentrations seen in smokers, also suppresses the immune system, and the suppression is only partially attenuated by ganglionic blockers. Indeed, T cells express α7-nAChRs; however, in spite of near total homology with the neuronal α7-nAChR, the T cell α7-nAChRs do not form detectable levels of ligand-gated Ca 2+ channels and operate essentially through second messengers. In T cells, nicotine modulates two distinct signaling pathways: 1. Nicotine increase [Ca 2+ ] i through the Src-like protein tyrosine kinase, Lck that requires the functional integrity of the T cell receptor. Nicotine affects NFκB activation through ERK that is independent of TCR. Thus nicotine modulates T cell and inflammatory responses through the sympathetic autonomic nervous system as well as via direct interaction with a unique α7-nAChR. Thus, nicotine controls the adaptive and inflammatory responses through sans and direct interaction with T cells via two distinct signaling pathways. PP08-12 Investigation of sensory neurogenic components in bleomycininduced scleroderma model using TRPV1 receptor and CGRP knockout mice Capsaicin-sensitive, transient receptor potential vanilloid 1 (TRPV1) receptor-expressing, sensory nerve terminals exert efferent activities besides the afferent function (nociception). This study examined the role of TRPV1 receptors and, calcitonin gene-related peptide (CGRP) in bleomycin-induced scleroderma using transgenic mice.Cutaneous sclerosis of TRPV1 receptor and calcitonin gene-related peptide knockout (TRPV1 −/− , CGRP −/− ) mice and wild-type (WT) counterparts was induced by daily s.c. injection of bleomycin throughout 30 days. Control groups were treated with the solvent (PBS). The excised skin samples were investigated with histological and biochemical methods. Composite cutaneous sclerosis score was calculated on the basis of thickening, leukocyte infiltration and amount of collagen bundles. The collagen-specific amino-acid, hydroxyproline, from the skin patches was measured with spectrophotometry. Quantitative real-time RT-PCR assay was used to determine type I collagen-alpha mRNA.Bleomycin treatment induced marked cutaneous thickening and fibrosis compared to the PBS-treated group. Composite sclerosis score was 14.5% greater in TRPV1 −/− and 43.1% greater in CGRP −/− mice compared to the respective WT groups. Similarly, the amount of hydroxyproline increased by 65.0% in TRPV1 −/− and by 25.3% in CGRP −/− animals versus the WT group. Type I collagen-alpha mRNA content was not significantly higher in the skin of bleomycin-treated TRPV1 −/− compared to TRPV +/+ mice on the 28th day of the experiment.These results suggest that activation of TRPV1 receptors by inflammatory mediators induces the release of sensory neuropeptides, which exert protective action against fibrosis.Grants: OTKA T046729, ETT-05-598/2003, RET-008/2005, The Wellcome Trust. PP08-13 Immunological and neurological roles of 80-kDa and 100-kDa haemopoietic factors Medicine, Allied Health Sciences, Kitasato University, Sagamihara Japan; c Institute for Animal Reproduction, Kasumigaura, Japan; d Japan Red Cross Hospital, Tokyo, Japan 80-kDa and 100-kDa factors were originally detected in the conditioned medium of cloned rat thymic myoid cells. Both factors commonly induce the growth and differentiation of monocytic lineage cells from the peritoneal, bone marrow, thymus and brain. Colonies induced by the two factors were different in morphology in an early stage of culture, but showed monocytic cell characteristics in the terminal stage. Immunohistochemical studies indicated that major producers of the two factors were neural cells in the brain and myoid cells in the thymus, suggesting their biological roles other than monocytic cell stimulatory activity. To examine biological roles, such as immunological and neurological roles, we purified those factors from the brain cells, cultured myoid cells and hyperplastic thymus in which many precursor myoid cells were detected, and tested their immunological and neuro-stimulatory roles. We found that those two factors sustain neuronal growth and stimulate microglial cell growth. Furthermore, the factors stimulate B-cell responses in a synergic fashion with IL-2, without the aid of other T-cell factors such as IL-4 or T-cells themselves and induce immunoglobulin class switches. Those factors also appeared to stimulate T-cells to produce B-cell activating cytokines, leading to B-cell proliferation. These results suggest that the two new haemopoietic factors play important roles not only in monocytic-microglial cell diversification but also in brain physiology and many immunological events. The proinflammatory cytokine interleukin-1 (IL-1) is produced both in the periphery and the brain in response to various immune challenges, including autoimmune diseases, as well as following exposure to psychological stressors. Since in humans autoimmune diseases and chronic stress are important risk factors for the development of major depression, and because our previous work implicated IL-1 in behavioral suppression following acute challenges, we sought to examine the role of this cytokine in mediating the depressive-like symptoms induced during episodes of experimental autoimmune encephalomyelitis (EAE) as we well as following exposure to chronic mild stress (CMS), an established model of depression in rodents. Using mice with deletion of the IL-1 type I receptor or transgenic over-expression of IL-1 receptor antagonist (IL-1ra) in the brain, as well as pharmacological administration of IL-1ra, we demonstrated that the depressive-like effects exhibited by mice with EAE or following exposure to CMS were completely abolished by blockade of IL-1 signaling. Furthermore, we showed that the behavioral effects of CMSinduced elevation of IL-1 are mediated both by adrenocortical activation and via suppression of hippocampal neurogenesis. These findings indicate that IL-1 is both necessary and sufficient for the depression associated with autoimmune and chronic stress conditions. PP09-02 Neural correlates of IgE-mediated allergy Although many authors had considered a direct interaction between allergic reactions and behavioral changes, supporting evidence had been elusive. In this series of studies we show that after oral or nasal ovalbumin (OVA) challenge, allergic mice present increased Fos-expression in the paraventricular nucleus of the hypothalamus (PVN) and in the central nucleus of the amygdala (CeA); mice with food allergy display higher levels of anxiety and had increased serum corticosterone levels; and allergyactivated neurons express corticotropin-releasing hormone in the PVN and CeA. OVA-allergic mice develop aversion to an antigen-containing solution, and also avoid a dark compartment previously associated with nebulized OVA. Results on brain Fos expression and behavioral data seem compatible with adaptive responses. Removing IgE by either antibody depletion or the development of oral tolerance precluded every response analyzed here. Csensitive fiber destruction by neonatal capsaicin inhibited the activation in the PVN, but not in the CeA, and decreased the magnitude of food aversion. Cromolyn, a mast cell stabilizer, completely blocked Fos expression in the PVN and CeA, and precluded the development of aversion to the dark compartment associated with nebulized OVA. Employing mice that do not develop an important inflammatory infiltrate following nasal OVA challenge, we found that inflammatory cells are not required at the site of challenge in order to trigger neural or behavioral correlates of murine experimental asthma. Altogether, we build a solid ground for understanding neuroimmune interactions during allergic responses that may contribute to the comprehension of psychological disorders associated with allergy. PP09-03 Interleukin-18 induces microglial activation in acute stress A growing number of researches report that microglial cells contribute to progress of neurodegeneration, such as Parkinson, Alzheimer, HIV encephalitis and multiple sclerosis. In the present study we demonstrated stressinduced microglial activation in the brain. Based on the finding that circulating IL-18 levels are elevated during the stress, we hypothesized that IL-18 may be involved in the microglial activation in the brain. To test this hypothesis, we administered recombinant IL-18 and monitored microglial activation 6 h after the administration both in-vivo and in-vitro. Under the control conditions as well as 1 ng/g intraperitoneal administration of IL-18, no significant microglial activation was observed. At 5 ng/g of intraperitoneal IL-18 administration, significant microglial activation started to occur. Clear microglial activation was observed following the administration of IL-18 of 10, 20 ng/g as well. In addition, IL-6 and iNOS were induced in microglial cells in response to IL-18 administration. Quantitative real-time PCR study revealed that IL-18 administration induced IL-6, IL-18, iNOS mRNA in cultured microglial cell lines in a dose dependent manner. Furthermore, in IL-18 null mice, microglial activation was reduced in the brain in response to 2 h period of stress. Thus, the present study demonstrates that circulating IL-18 may activate microglial cells in the brain, suggesting that stress may induce microglial activation through stress-induced IL-18. PP09-04 Age-related bias in function of natural killer T cells and granulocytes after stress: Reciprocal association of steroid hormones and sympathetic nerves Stress-associated immune responses were compared between young (8 weeks) and old (56 weeks) mice. Since stress suppresses the conventional immune system (i.e. T and B cells) but inversely activates the primordial immune system (i.e. extrathymic T cells, NKT cells, and granulocytes), these parameters were analysed after restraint stress for 24 h. The thymus became atrophic as a function of age, and an age-related increase in the number of lymphocytes was seen in the liver. Although the number of lymphocytes in both the thymus and liver decreased, the magnitude was much more prominent in the thymus. To determine stress-resistant lymphocyte subsets, two-color immunofluorescence tests were conducted. NKT cells were found to be such cells in the liver of young mice. On the other hand, an infiltration of granulocytes due to stress was more prominent in the liver of old mice than in young mice. Liver injury as a result of stress was prominent in young mice. This age-related bias in the function of NKT cells and granulocytes seemed to be associated with a difference in the responses of catecholamines (high in old mice) and corticosterone (high in young mice) after stress. Indeed, an injection of adrenaline mainly induced the infiltration of granulocytes while that of cortisol activated NKT cells. These results suggest the existence of age-related bias in the function of NKT cells and granulocytes after stress and that such bias might be produced by different responses of sympathetic nerves and steroid hormones between young and old mice. PP09-05 Salivary cortisol and DHEA response to psychosocial stress in socially anxious individuals Social Anxiety Disorder (SAD) is the most common type of anxiety disorders. Kessler et al. SAD often interfere their academic, social, and occupational function. SAD is characterized by the fear of negative evaluation by others, so the patients feel anxiety too much in socially evaluative situation and it impairs psychological and physiological states. In the present study, we conducted Trier Social Stress Test (TSST; Kirschbaum et al., 1993) , which produces firm evaluative situation, to investigate salivary cortisol and dehydroepiandrosterone (DHEA) response in socially anxious people. This study was approved by the Ethical Committee of Waseda University. The participants were 17 male students, divided into 9 high socially anxious (SA) group and 8 low SA group by the score of Short Fear of Negative Evaluation Scale (SFNE; Sasagawa et al., 2004) . Before experiment, they were informed the purpose of present study and signed the consent form. All experiments were conducted between 15:00 and 19:00. In the results of this study, significant correlations were found between AUC (Area Under the Curve) of cortisol and SFNE (r = − .45, p = .07), and between AUC of cortisol/DHEA ratio and SFNE (r = − 56, p = .02). Further analysis showed DHEA in high SA group rose greater than in low SA group. On the other hand, cortisol in high SA group reacted less in TSST. These results indicated that there was endocrine imbalance in socially anxious people to social stressor and this imbalance might affect the maintenance of SAD. PP09-06 Appraisal about controllability of acute stressor and braincardiac-immune association Acute stress elicits multiple responses in autonomic, endocrine, and immune systems. Cognitive appraisal has been thought as one of important modulators of such stress responses. To investigate brain substrates of such crosstalk between the homeostasis systems accompanying cognitive appraisal of an acute stressor, we recorded simultaneously regional cerebral blood flow (rCBF) with 15 O-water positron emission tomography, cardiovascular indices (heart rate (HR) and blood pressure (BP)), neuroendocrine indices (concentration of epinephrine, norepinephrine, and adrenocorticotropic hormone (ACTH) in blood), and immune indices (proportions of subsets of lymphocytes (NK cell, helper T cell, cytotoxic T cell, and B cell) in blood) when 11 male subjects conducted a mental arithmetic task with high controllability (HC) and low controllability (LC).The LC task caused less sense of control for subjects than the HC task. Significant increase of rCBF in the medial and lateral orbitofrontal cortex (OFC) and medial prefrontal cortex (MPFC) was observed in a comparison of the LC minus the HC tasks. More importantly, significant positive correlations between rCBF and the HR, BP, and NK cells were found commonly in the lateral OFC in the LC task, but not in the HC task. The present result showed for the first time that the OFC might be one of pivotal regions for top-down regulation over cardiovascular and immune activity accompanying cognitive appraisal on an acute stressor. PP09-07 Salivary secretory Immunoglobulin A modulation induced by repetitive stressful task and the mathematical model of the modulation S. Nomura Department of Mathematics and Computer Science of Shimane University, Matsue, Japan Recent psychoneuroimmunological and psychoendocrinological studies revealed that the human immune and endocrine secretory substances change its levels sensitively along with participants' subjective mental stress and relaxation. However, the precise modulation of those substances induced by such a mental interventions and its mechanism are still unknown. In this study, one of the major secretory immune substance, secretory Immunoglobulin A (sIgA), was assessed to investigate the normal human secretory response induced by a stressful task of deskwork operations focusing on following two points: (1) the precise changing of salivary sIgA under the repetitive short-term stressful works with beaks procedure, and (2) the mathematical model of sIgA modulation induced by that procedure. The results of the experiment in this study indicated that the changes of sIgA concentration within saliva clearly reflected the participants' subjective stress level with a certain time delay depending on the duration of stressful work. Referring with the results of the experiment and previous sIgA studies, I then propose a mathematical model of sIgA modulation based on the logistic function, which is the nonlinear ordinary differential equation consisting of the first order of exponential increasing term and the second order of nonlinear decreasing term, and in other words, it possesses the homeostatic property of living organisms. The result of the simulation of the model is well consistent with the sIgA modulation profiles of the experiment. Finally, I introduce a blue print of the deskwork stress prediction system constructed via the mathematical model. PP09-08 Oxidation detected by ultra-weak chemiluminescence may be related to neural-immune interactions under acute psychological stress Aim: Stress sensed by the neural system affects the immune system. Previous studies described elevations of secretory immunoglobulin A (sIgA) levels in saliva and increased oxidative status when exposed to acute psychological stress. Human saliva contains peroxidase and the electron donor SCN-. Ultraweak chemiluminescence (UCL) is based mainly on an electronic transfer in the oxidation-reduction reaction and it registers the oxidative reaction. In order to investigate the relationships between immune functions, oxidative stress and neural reactions, the effects of performance anxiety on salivary UCL were examined and the values were compared with the salivary sIgA levels. Participants and methods: Participants were 10 volunteer students. The subjects performed a task of a serial aseptic manipulation for cell culture. Saliva was collected 30 min before, immediately after, 30 min after and 60 min after the performance. Results: One-way repeated measures ANOVA for UCL revealed a significant difference across the four measurement occasions (F =4.45, p< 0.05). Post hoc Dunnett's test clarified that UCL levels immediately after the task was significantly greater compared with that 30 min before (p< 0.05). Two-way ANOVA with repeated measures indicated that UCL and sIgA exhibited the same change pattern in the four occasions (F= 0.28, p= 0.84). Conclusion: UCL counts a volume of photons generated during oxidative reactions, which can be an effective monitor of lipid peroxide generation. It was suggested that oxidation may occur during the neural-immune interactions. PP09-09 Therapeutic effects of color on acute psychological stress Kuniaki Takagi 1 , Nobuhiro Goi 1 , Yuko Hirai 1 , Hitoshi Harada 1 , Akira Ikari 1 , Kimitsugu Nakamura 2 , Mitsuo Hiramatsu 2 , Hirohito Tsuboi 3 , Reiko Horiki 4 , Takahiko Ono 1 and Naohide Kinae 1 1 University of Shizuoka, Shizuoka, Japan; 2 Hamamatsu Photonics K.K., Hamamatsu, Japan; 3 University of Hamamatsu, Hamamatsu; 4 Color Works, Tokyo, Japan Aim: Thalassotherapy is used for various diseases from the age of Hippocrates; however, the effective factor and the mechanism are almost unknown. In this study, we focused attention on a blue that was the color of the sea, and examined the influence of the color on acute psychological stress. Participants and methods: Participants were 137 volunteer students. The subjects were divided into eight groups, blue to investigate the color effects. The subjects of four groups had worn color glasses before the Kraepelin test which was acute psychological stress. On the other hand, the subjects of the other groups had worn color glasses after the Kraepelin test. The saliva was collected from the subjects in each stage and the stress markers were measured. Results: In the subjects who had worn color glasses before the test, the salivary IgA (sIgA) and ultraweak Chemiluminescence (UCL) of the subjects in the Red and the Green were higher than that of the group of The Blue. Additionally, in the subjects who had worn color glasses after the test, the sIgA and UCL of the subjects in the Blue and the Green were significantly lower than that of the group of The Red. Conclusion: From these results, we suppose that there is a calming and a recovery effect from an acute psychological stress in blue. PP09-10 High motor active rats in novel environment (HR) compared to low motor active (LR) rats are not better against cancer M.K. Jurkowski, B. Bobek-Billewicz Institute Of Oncology, Gliwice Branch, Poland HR and LR rats show a strong correlation between the motility and psychostimulants' self-administration and the differences in their locomotor activity are an index of their behavioral reactivity to the stressful situation. The aim of the study was to asses HR and LR natural killer cell cytotoxicity (NKCC) and, while sometimes significant changes in the concentration and functional activity of immune parameters observed not necessarily lead to higher incidence of illness, we also assessed in vivo HR and LR rats' susceptibility to solid tumor carcinogenesis.The peripheral blood natural killer cell cytotoxicity (NKCC) (standard 4 h 51 Cr release assay against YAC-1 target cells, E:T = 50:1; 25:1; 12:1), cytotoxicity of NK lymphocytes at the single cell level (agarose assay) and chemically induced tumor incidence and tumor growth rate were measured.The results indicated significant (≈ 20%, p < 0.001) differences in NKCC between HR (n = 45) and LR (=45) rats (E:T = 50:1; 33.4 ± 0.8% vs. 27.0 ± 0.8%, E:T = 25:1; 22.4 ± 0.6% vs. 18.0 ± 0.5%, E:T = 12:1; 13.7 ± 0.6% vs. 10.9 ± 0.5%) respectively. Evaluation of a single cell lytic ability on the same effector cells did not reveal any significant effect on the percentage of conjugate formation and on the target lysis. There were no significant differences either in tumor incidence or in tumor growth rate also.The results suggest that HR rat's higher NKCC may not necessarily lead to diminished incidence of carcinogenesis or LR rats posses more effective other anticancer mechanisms. PP09-11 Effect of attraction to the favorite person on innate immune system Everybody can "fall in love". Thus everybody knows that attraction to the favorite person invokes positive feelings and often makes us energetic. The question arising here is whether biological systems, such as autonomic nervous, endocrine, and immune systems, are affected by this positive emotion. To investigate effects of attraction to the favorite person on the biological systems, we recorded various parameters, namely mood states, heart rate (HR), skin conductance level (SCL), serum levels of several hormones (endorphin, oxytocin, vasopressin, norepinephrine, epinephrine, and cortisol), and proportions of T cells and natural killer (NK) cells in the lymphocytes simultaneously when subjects viewed the video films of their favorite persons. The mood states were assessed by the profile of mood status (POMS), the serum levels of several hormones were measured by Enzyme-Linked Immunosorbent Assay (ELISA), and the proportions of T cells and NK cells in the lymphocytes were measured by flowcytometer. As results, accompanying self-reported vigorous and better feeling, SCL and the proportion of NK cells significantly increased. No significant changes in HR, the serum levels of various hormones, as well as the proportion of T cells in the lymphocytes were found. These results suggest the possibility that attraction to the favorite person may have a role in activating NK cellrelated innate immune system by means of the activation of SCL-related autonomic nervous system. PP09-12 Temporal redistribution of NK cell subsets during acute psychological stressor K. Kimura a,b , T. Isowa a , and H. Ohira a a Department of Psychology, Nagoya University, Aichi, Japan; b Japan Society for the Promotion of Science There were accumulated evidences indicating that an acute psychological stressor could elicit lymphocyte redistribution via sympathetic nervous activation. Furthermore, it was also known that a temporal variation of this redistribution was different between lymphocyte subsets. For instance, under the acute stressor, CD3 − CD16 + CD56 + natural killer cells (NK cells) were remarkably increased in peripheral blood while CD3 + CD4 + helper T cells were slightly decreased or showed no change. One possible explanation about the distinct variations was adhesion molecules expressed on the surface of lymphocytes such as CD62L and CD11a. Recent researches suggested the redistribution of NK cells was occurred differentially based on CD62L expression density. And distinct CD62L expression in NK cell subsets was corresponded to functional differentiations, namely CD56 +high immunomodulatory function and CD56 +low cytotoxic function. Although differential redistributions of these subsets were reported by several studies, temporal variation of these subsets and the relationship with sympathetic nervous activation remained unclear. Thus, present study was conducted to examine these issues in acute stressor paradigm. Ten human participants performed mental arithmetic task as an acute stressor and their blood samples for estimating CD62L + or CD62L-NK cells, CD62L + or CD62Lhelper T cells, epinephrine, norepinephrine, and cortisol were taken at each 2 min during the task. Additionally, blood pressure and heart rate were monitored during the experiment. As a result, NK cells showed remarkable increment by the acute stressor and these variations indicated the close relationship with sympathetic nervous activation such as elevated blood pressure. PP09-13 Psycho-educational intervention for perceived social support has an effect on Natural killer cell activity among the elderly in rural Japan Background: Relatively little research showed the strategies to change perceived social support to improve health outcomes. The previous intervention study for the elderly women focused on giving enacted social support, and failed to improve perceived social support. So we conducted psycho-educational intervention for the improvement of attitudinal perceived social support among general elderly people and examined the effect on natural killer cell activity. Method: A randomized control cross-over design involved 60 participants 65 years old and over. A ninety-minute intervention was conducted weekly for five successive weeks. The intervention consisted of (1) a lecture about illness and its prevention, (2) relaxation using the 'Dohsa' therapy, and (3) the psycho-educational intervention for perceived social support. Each participant was administered questionnaires and taken a blood sample between 9:00 am and 11:00 noon to control for diurnal variations at four times (baseline, pre-intervention, post-intervention, and follow up). NK cell activity was measured against K562 using a standard 4-h51Cr release assay. Result: There was a significant improvement in the perceived social support score and natural killer cell activity only in the intervention group at postintervention period and follow up period. Conclusions: We found that the perceived social support score could be increased by this psycho-educational intervention. It was also suggested that the change of perceived social support had a causal effect on the immunity such as natural killer cell activity. PP09-14 Effect of cytokines and neurosteroids on neuronal cell death of Neuro2A in response to NaF Ariyuki Kagaya 1 , Minoru Takebayashi 2 , Shigeru Morinobu 3 , and Shigeto Yamawaki 3Recent progress in neuroscience shows that various neurotransmitters, hormones and cytokines play essential roles in neuronal cell survival and death. Various kinds of stress elicit cellular responses, leading to cell death. Sodium fluoride (NaF) is a well-known chemical stressor, and recent reports suggest that NaF induces cell death. In this study, we have investigated the effect of cytokines and neurosteroids on NaF-induced cell death of Neuro2A.Neuro2A cells were cultured in DMEM containing 5% FCS on 96-well microplates. The cells were then maintained without or with sodium fluoride (NaF) and/or other indicated drugs. After the cells were treated with the drugs, they were incubated with WST-8 to measure cell viability. Then, the optical density at 450 nm wavelength was measured with a microtiter plate reader.NaF induced cell death of Neuro2A in its dose and time dependent manners. IL-1alpha, IL-6, TNF-alpha, or LIF had little effect on the cell survival. DHEA, or DHEAS had no effect on the cell survival. These compounds had little effect on NaF-elicited cell death.However, estradiol significantly inhibited the cell death induced by NaF. These results suggest that estradiol has an effect to protect Neuro2A against NaF-induced cell death. PP09-15 Cytokines modulate neurotrophin-dependent axon outgrowth of dorsal root ganglia -Dose-dependent effects of IL-4, IL-6, IFNg and TNFa Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité -Universitätsmedizin Berlin, Germany Inflammation is part of the wound healing response after mechanical lesion of the peripheral nervous system. However, it is not clear whether inflammatory cytokines exert a beneficial or detrimental effect on neurotrophin (NT)-dependent axonal outgrowth of dorsal root ganglia (DRG). Here, we have analyzed the effects of NTs and cytokines on neurite outgrowth of murine embryonic DRGs in vitro. DRGs were cultured in Matrigel with NGF, NT3 and NT4. All NTs (50 ng/ml) were applied alone, in double or in triple combinations. IL1b, IL4, IL6, IFNg and TNFa (50 or 500 ng/ml) were added to the different NT combinations. Using an image analysis system, the neurite outgrowth zone and axonal density were determined after 48 h. NT3 and NT4 alone and in combination induce a moderate increase in axonal growth, while NGF and NGF-including combinations substantially increase axon growth. TNFa inhibited NT3 − , NT3 + NGF − , NT4 + NGF − , and NT3 + NT4 + NGF-induced outgrowth.In summary, NTs differentially stimulate DRG axonal outgrowth and density. This NT-induced axon growth is dose-dependently modulated by cytokines. PP09-16 Diazepam effects on blood neutrophil activity and on Ehrlich tumor growth Introduction: Benzodiazepines, such as diazepam, are commonly used for their anxiolytic effects, i.e., by their action on high affinity receptor sites coupled to GABA A receptor complex. Diazepam treatment leads to an increase in serum corticosterone levels, which correlates positively to immunity alterations in laboratory animals.Objective: To analyze diazepam effects on innate immunity and on resistance to Ehrlich tumor growth. Methods and results: Swiss Male mice were divided in two groups. Animals from experimental group (E) and control group (C) were treated, per oral, with diazepam (3.0 mg/kg/day) or with control solution (0.1 ml/10 g/day) for 7 days, respectively. 5.0 × 10 6 Ehrlich tumor cells. Blood neutrophils activity and tumor growth were evaluated after 7 days. Animals treated with diazepam presented: 1a decrease on basal neutrophil oxidative burst (C = 36.56 ± 8.60; E = 28.05 ± 7.11); 2an increase on neutrophil oxidative burst after an in vitro induction with phorbol-miristate-acetate (C = 83.72 ± 15.88; E = 109.97 ± 22.57) as well as with Zymosan (C = 113.83 ± 21.18; E = 154.01 ± 34.02); 3an increase of both neutrophil intensity (C = 25.87 ± 13.31; E = 42.93 ± 11.64) and percent of Zymosan phagocytosis (C = 77.15 ± 9.30; E = 87.40 ± 6.83); 4an increase on Ehrlich tumor growth as evaluated by total number of tumor cells of ascitic fluid (C = 58.16 ± 14.00; E = 78.30 ± 19.17 × 10 7 ). Conclusion: Diazepam changes neutrophil activity measured in vitro and decreases animals resistance to Ehrlich tumor growth. PP09-17 The effect of atypical antipsychotic drugs on the production of proinflammatory cytokines, nitric oxide and reactive oxygen species by activated microglia Microglia has recently been regarded to be a mediator of neuroinflammation by releasing proinflammatory cytokines, nitric oxide and reactive oxygen species (ROS). Microglia thus plays an important role in the pathology of neurodegenerative disease such as Alzheimer's disease (AD) and Parkinson's disease (PD). The pathological mechanisms of schizophrenia remain unclear while some recent neuroimaging studies suggest even schizophrenia may be a kind of neurodegenerative disease like AD and PD. There have so far been only a few studies on the relationship between microglia and schizophrenia. Atypical antipsychotic drugs (APDs) are becoming standard drugs for the treatment of schizophrenia because of their less adverse effects and more effectiveness for the negative symptoms of schizophrenia. We thus investigate the effects of atypical APDs on the production of proinflammatory cytokines, nitric oxide and ROS by activated microglia, and also try to clarify the molecular mechanisms underlying these effects. As compared with haloperidol, a typical conventional APD, several kinds of atypical APDs significantly inhibited the production of proinflammatory cytokines such as IL-1β, IL-6 and tumour necrosis factor α (TNF-α), and nitric oxide by lipopolysaccharide (LPS) + Interferon-γ stimulated microglia. These results suggest that atypical APDs may have more beneficial effects on proinflammatory conditions in the CNS which may play a crucial role in the pathology of schizophrenia. PP09-18 Lithium chloride modulate functions of human monocytederived dendritic cell For almost half a century, lithium has been one of the most widely used medications for bipolar depressive illness, although its therapeutic mechanism of action remains obscure. Accumulated preclinical and clinical evidences for the action of lithium in the brain suggest that lithium can affect membrane transport systems, neurotransmitter receptor regulation, second messenger systems, protein kinase C (PKC) regulation, and gene expression. Recent studies showed long term, but not acute, treatment of cultured cerebellar granule cells with LiCl induce a concentration-dependent decrease in proapoptotic p53 and Bax level; and remarkably increased cytoprotective Bcl-2 protein. These results suggest that lithium, in addition to its use in the treatment of bipolar depressive illness, may have an expanded use in improving neurodegeneration. However, regulation of lithium on the dendritic cell was not-well defined. Dendritic cells (DC) are key regulators of immune responses, and were involved in the initiation and maintenance of both innate and the adaptive immune responses. We demonstrate lithium chloride treatment enhanced human monocyte-derived CD86 expression. In addition, lithium chloride increased cytokines, TNF-a IL-8, IL-1b, IL-10 and IL-6 production. However, lithium have no effect on T cell proliferation which assayed by mix lymphocyte reaction. These data suggest that lithium chloride might modulate DC functions via phenotype and cytokines expression. PP09-19 Association of polymorphism in the IL-1ra gene with autism and pervasive developmental disorders Over the last decade it became evident that pervasive developmental disorders (PDD) in general, and autistic disorder more specifically, are associated with several immune alterations and autoimmune processes. In particular, the innate immune system seems to be activated, accompanied by the production of pro-inflammatory cytokines and neuroinflammatory processes. Viruses, such as measles, and environmental toxins, such as mercury, which are known stimulators of innate immunity have been implicated in the etiology of autism. These stimuli may interact with genetic variables, as there is an increased prevalence of familial autoimmune disorders in parents of probands with PDD. The pro-inflammatory cytokine interleukin-1 (IL-1) may be particularly relevant to PDD because (1) it is involved in emotional, social and cognitive modulation, (2) it is induced by vaccination and toxins, (3) it mediates the immune responses to mercury, and (4) it plays a major role in autoimmunity. Recently, a variable number tandem repeat polymorphism in the second intron of the IL-1 receptor antagonist (IL-1ra) gene has been found to be associated with increased risks of chronic inflammatory and autoimmune diseases. To test the hypothesis that this polymorphism is associated with autism we are currently examining 116 families (128 probands diagnosed with the ADI-R and ADOS-G, and their parents) using a family-based association test (UNPHASED). Furthermore, we are assessing whether the association with IL-1ra is mediated by the role of this gene in specific disturbances in socialemotional and cognitive parameters. The results may implicate genetic alterations in IL-1 signaling in autism and its relationship with autoimmune processes. Major Depression Disorder (MDD) is a common with prevalence of about 15% for men and up to 25% in women. In recent years, the association of immune system alteration and MDD has been reported on the basis of new observations. Therefore, we decided to do more accurate assessment in determining or excluding the relationship between immune system and MDD. Materials and methods: In patients and control group, we determined TNF α, FN γ, IL-4 and IL-10 (by ELISA), serum immunoglobulin, serum proteins, and markers of immune system cells include CD 16-56, CD19, CD 8, CD 4, CD 3. Results: This study was include 37 patients (28/9: F/M) with mean age of 39 years and fifteen number control. In cytokines study were observed no significant differences between the patient and control group. In the patient group (moderate grade), there was significant relationship only between IL-10 and IFN γ (p = 0.04). The amount of total protein and γ in 95% of the patient group were higher than normal range and the number of NK, B, and T cells was in the normal range. Discussion: In the patients with MDD, inhibitory cytokine (IL-10) was increased in severe grade of disease.Total protein of patient group was higher than normal range but immune system cells and Immunoglobulin were in the normal range. PP09-21 Relevance of cytokines, chemokines and other immune mediators for immunomodulatory strategies in patients suffering from chronic idiopathic pain syndromes This is the first study to give insight to a wide pattern of chemokines, cytokines and their receptors, as well as corticotropin-releasing factor (CRF), substance P (SP) and histamine in serum and cerebrospinal fluid of patients suffering from uncontrollable chronic idiopathic pain syndromes compared to controls. Patients and methods: 10 patients suffering from idiopathic chronic widespread pain syndromes (CWP), such as Fibromyalgia and Myofascial Pain Syndrome (in some cases additionally Complex Regional Pain Syndrome), all of them refractory to conventional pain treatment strategies and 9 controls who underwent spinal anaesthesia for minor lower extremity surgery were enrolled in this study. Serum and cerebrospinal fluid concentrations of above mentioned parameters were determined using high-sensitivity ELISA kits and Luminex-100 technology. Results: In patients' serum SP, histamine, IL-12(p40/p70), Eotaxin, MIP-1a, MIP-1b, MCP-1, IL-5, IL-6, MIG and IL-8 were significantly elevated compared to controls. In cerebrospinal fluid SP, CRF, IL-10, IL-1Ra, IFNa, MCP-1, IL-2, MIG and IL-8 were significantly higher compared to controls. G-CSF was detectable in cerebrospinal fluid of 9 patients but in none of the controls. However, IL-1b, RANTES, IL-13, IL-15, IL-7, GM-CSF, sIL-6R, sIL-2R, IL-4, Gro-a, MCP-3, TNFa, MCP-2, IL-7, IP-10 and GDNF were not detectable in any sample or did not differ significantly between patients and controls in both, serum and cerebrospinal fluid. Conclusion: Our findings indicate a possible role of immunopathological mechanisms in the development of therapy-refractory chronic pain. PP09-22 Increased NK activity in a group of schizophrenic patients Functional alterations in the immune system of schizophrenic patients have been shown in different studies. The importance of NK cell activity in schizophrenia is due to their role in immune surveillance against tumoral and viral cells. These studies aim to explain the low incidence of cancer in schizophrenic patients. But no consistent findings have emerged. The purpose of this study was to evaluate NK activity in schizophrenic patients with eliminating the effect of such factors. In this study, we have chosen 30 medication-free schizophrenic patients and 41 healthy sex, age and smoking status matched individuals. NK cell activity of case and control subjects was measured by MTT (Methyl-Thiazol-Tetrazolium) test. Statistical analysis showed a significant increase in NK activity in schizophrenic patients compared with controls (P = 0.011). Also, NK activity of smoking patients was significantly lower than that of nonsmoking patients (P = 0.02). Other demographic factors did not show any influence on NK activity. The higher activity of NK cells in the schizophrenic patients as compared with the control population could explain the low incidence of cancer in these patients. And decreasing effect of smoking on NK activity in the patients could be one of the responsible factors for the inconsistency in results of different studies. For decades, the relationship between the Immune system and schizophrenia has attracted considerable research in Psychoneuroimmunology (PNI). There are a few etiologic hypotheses connected to this proposed relationship, being Viral infections, Autoimmunity and Activation of Tlymphocyte-Macrophage. This Study focuses on a comparative analysis of the immune function of 30 drug-free schizophrenic patients and 42 healthy individuals who are matched in sex and age and Smoking status. In this analysis, proliferation of T cells in response to PHA (PhytoHaemAgglutinin) mitogen, was measured with MethylThiazolTetrazolium Test (MTT). Serum ANA (Anti-Nuclear-Antibody) level and ACA (AntiCytoplasmic-Antibody) presence and Circulating Immune Complexes (CIC) concentration were measured with Indirect Immuno-Fluorescence and PEG precipitation tests, respectively. The results of these assays were then analyzed with SPSS 11.5. T cell proliferation of patients had a mean of 1.96417 ± 0.830684 and T cell proliferation mean of normal subjects was 3.72971 ± 1.398237. Statistical analysis showed a significant decrease in T cell proliferation in schizophrenic patients compared with controls (P = 0.000). Serum ANA level and CIC concentration were not significantly different between the two groups (P = 0.661, P = 0.102, respectively). These results could be evidences of an Autoimmune/Viral hypothesis in at least a group of schizophrenic patients. PP09-24 Association between interleukin-10 promoter polymorphisms and schizophrenia in Iranian population Several reports show abnormal cytokine levels in psychotic patients and indicate a possible role of the immune response system in the pathogenesis of schizophrenia. Interleukin-10 (IL-10) gene maps on chromosome 1(q31-q32), a locus associated with genetic susceptibility to schizophrenia. To investigate the role of IL-10 on schizophrenia, we analysed the association between four single nucleotide polymorphisms (SNPs) located on IL-10 promoter (−592A/ C, −1082A/G, −2763C/A, −3573T/A) and the risk of schizophrenia in patients (n = 321) and unrelated controls (n = 392), all residing in the city of Mashhad, Iran. The frequencies of IL-10 promoter-3575 T/A SNP T allele and IL-10 promoter −2763C/A SNP C allele were also significantly increased in patients compared with controls (p < 0.05). Furthermore, comparing to controls, the frequency of IL-10 −1082 A allele was significantly increased in all patients (p =0.024), patients with positive family history (n = 63) (p = 0.015), undifferentiated (p = 0.031), non-disorganized (p = 0.011) and nonresidual (p = 0.02) subtypes. IL-10 promoter −1082 AA genotype was significantly increased in family history positive patients (p =0.021) and nondisorganized groups. Our preliminary haplotype analyses suggest that the frequency of IL-10 promoter haplotypes, which are associated with high IL-10 production are increased in schizophrenia. Our results suggest that IL-10 promoter SNPs may be associated with the increased risk of schizophrenia. This study shows the different genetics background of schizophrenia subtypes in Iranian population. PP09-25 Chronic exposure to vapors of gasoline and diesel upregulates the metallothionein I and II expression in the olfactory brain of mice Olfactory systems are often used as excellent models for the study of widespread aspects of neural development and neuron-glia interactions after exposure to agents whose route of entry into the CNS might bypass the bloodbrain barrier. Since, these pathways might be frequently stimulated by airborne urban pollutants resulting in chronic diseases, in this study we attempted to evaluate the effects of intermittent exposure of mice to gasoline (G) and diesel (D) vapor on brain, lungs and kidney expression of metallothioneins I + II (MTs), which as cysteine-rich proteins with metal binding affinity might provide cytoprotective action on any form of stress or injury.For this purpose C57/BL6 mice were for 1 h/day closed in a small metabolic chamber ventilated with fresh air or with G or D vapors. Protocol was repeated for 10 days and then MTs expression was detected in the brain, lungs and kidney. The findings were compared with data obtained in unstressed mice. It was found that G and D inhalation markedly upregulated the MT expression in ventricular ependyma and astrocytes, and particularly in the hippocampus, where project the stellate and pyramidal cells of lateral olfactory tract and perforant path. Simultaneously, both stress and toxic agents increased the cytotoxicity of hepatic and splenic lymphatic cells against the syngeneic thymocytes, implying that autoreactive clones of cells participate in the processes of tissue repair (supported by grants from Croatian Ministry of Science). PP09-26 Modulation of pro-inflammatory and neural activity-related gene expression in the olfactory bulb of mice by nasal inhalation of low-level toluene Sohel Ahmed, Hidekazu Fujimaki, Tin-Tin-Win-Shwe, Shoji Yamamoto, Shinji Tsukahara, Yoshika Kurokawa, Daisuke Nakajima, and Sumio Goto National Institute for Environmental Studies, Ibaraki, JapanSince the induction of multiple chemical sensitivities might be related to volatile organic compounds in houses and offices, it is speculated that there is a positive relationship between immunological and neurological alterations and inhalation of volatile organic compounds. To study the effect of exposure to low-level toluene on the olfactory system neuroimmune responses, male C3H/HeN mice were exposed to filtered air (control), 9, and 90 ppm toluene in nose-only exposure chamber for 30 min per day, on days 0, 1, 2, 7, 14, 21, and 28. These mice were either maintained at non-immunized or immunized condition with ovalbumin (OVA). Using a quantitative real-time PCR method, we investigated the olfactory bulb expression of several genes. Our data demonstrate a significant difference of mRNA-expression of pro-inflammatory genes, interleukin-1β, CCL2 and CCL3 in the OVA-immunized mice exposed to 9 ppm toluene, as compared to that of filtered air control and 90 ppm toluene. In case of the neural activity-related genes, we observed a similar pattern for the glutamatergic NMDA receptor subunits, NR2A and NR2B. However, we did not observe any such pattern for the dopaminergic receptor subtypes, D1 and D2. This is the first study to show in vivo modulation of the olfactory bulb expression of pro-inflammatory and neural activity genes in mice by nasal inhalation exposure to low-level toluene, in concert with antigenic stimulation. PP09-27 Influence of emotion regulation on the immune, autonomic responses, and cognition H. Murakami, H. Ohira Psychology, Nagoya University, Aichi, JapanThere are some emotion regulation techniques to decrease facing anxiety and followed physiological responses. But there is an adaptive self-focus which decreases anxiety state. The former is maladaptive self-focus includes conceptual, analytical, evaluative way of self-focus. The later is adaptive focusing on direct, intuitive, experiential awareness of experience in the moment.Adaptive self-focus manipulation is known as one of the factor of the mindfulness.Mindfulness is the third wave cognitive behavior therapy which has been developed to treat a range of psychological disorders including depression, anxiety and fibromyalgia. We hypothesized that adaptive self-focus manipulation is also effective to decrease autonomic and immune responses. In cognitive theory, there is interaction between emotion and cognition. We also demonstrated the influence to cognition which affected by endocrine systems in anxiety state.Our purpose of this study is to demonstrate adaptive self-focus manipulation attenuates anxiety responses in the immune, autonomic responses, and cognitive negativity bias. PP09-28 Magnetic stimulation of left temporo-parieto-occipital (TPO) cerebral cortex: A non-invasive treatment to suppress ongoing human IgE responses We found that magnetic stimulation of left, but not right, TPO cerebral cortex of allergic humans significantly increased their blood CD4 + and CD8 + T cell, but not B cell, numbers at 4-6 h, and strongly suppressed their serum IgE levels at 4-5 days (>90%). In contrast, with right stimulation, the numbers of blood T cells decreased, and serum IgE levels did not change.We previously reported that electrical stimulation of rat left, but not right, TPO cortex increased cell export from the thymus, mediated by sympathetic pathways in the upper spinal cord, resulting in increased blood CD4 + and CD8 + T cells numbers at 4-6 h (Moshel et al. In contrast, with right stimulation, the numbers of blood T cells decreased. We are studying the ability of left vs. right electrical stimulation of TPO cortex to suppress peak hapten specific IgE responses of BPO-KLH sensitized rats, and the mechanisms involved.Supported by Research Foundation of SUNY. Multiple sclerosis (MS) is a chronic neurological disease affecting the central nervous system. The disease is characterized by demyelination and axonal loss caused by abnormal immunological responses resulting in accumulating neurological disabilities. MS is considered to be a complex disease where both genetic and environmental factors contribute to the pathogenesis.In this study we have investigated the genetic role of the CD4 gene in MS. Nine single nucleotide polymorphisms (SNPs) were selected for genotyping based on the LD pattern in HapMap and to get an even distribution of markers over the gene. 920 Swedish MS patients and 778 Swedish healthy controls were genotyped using the Sequenom method. No haplotype association was found and all associated SNPs were located within the same LD block. In order to confirm these results, all three significant markers were genotyped in an independent case control material, consisting of 1720 Nordic (Norway, Denmark and Finland) MS patients and 1416 Nordic healthy controls. None of the associations from the Swedish case/control material did replicate in the Nordic material. We conclude that the CD4 gene is not of major genetic importance in MS in a Nordic population.The study indicates two important factors in performing genetic association studies in complex diseases; the necessity of large case/control materials, and access to independent materials for confirmation of initial associations. PP10-02 Chromosome 19q13 and multiple sclerosis A. Bonetti University of Helsinki, Helsinki, Finland Previous studies have suggested a role of allelic variation on chromosome 19q13 in multiple sclerosis (MS) susceptibility. A marker in the 19q13.1 area showed nominally significant association with MS. This marker resides at a suggestive linkage peak, and associated with MS in a previous Italian study. Another association was found with an APOE haplotype on 19q13.2, supported also by a separate case-control analysis. Previous suggestions on ILT6 deficiency or association with D19S585 on 19q13.3-q13.4 were not replicated in this study.These results show that two subregions of chromosome 19q13 are associated with MS warranting further studies on their implication with the disease. PP10-03 Multiple sclerosis and the putative autoimmunity SNP CT60: An association study in patients from Germany, Hungary and Poland Polymorphisms in the CTLA4 gene region have been associated with susceptibility to autoimmune diseases (AID). The recently described single nucleotide polymorphism CT60, located in the 3′ untranslated region of CTLA4 has been shown to be associated with Graves' disease, autoimmune thyroiditis, autoimmune diabetes and other AID. Although one study showed an association of the +49A/G⁎G-CT60⁎G haplotype with Multiple sclerosis (MS), other studies failed to demonstrate association with CT60. Also, there is conflicting data regarding CT60 allele-dependent gene expression.We conducted an association study including so far a total of 630 patients from Hungary, Poland and Germany and 814 control persons. Although the CT60⁎G/G genotype was overrepresented among MS patients, this association did not reach statistical significance. We conclude, that the influence of CT60 on MS susceptibility isif at allrather small and might be restricted to subgroups of patients. Furthermore, we analyze genotype-dependent expression of CTLA-4 and ICOS on T cells after in vitro stimulation. PP10-04 Pro and anti-inflammatory cytokines gene polymorphism in Multiple Sclerosis patients Immunology Department, Tehran University of Medical Sciences, Tehran, Iran The role of inflammatory (IFN-γ, TNF-α, IL-6) and anti-inflammatory (TGF-β, IL-10) cytokines in the pathogenesis of Multiple sclerosis (MS) has been extensively studied. Elevated levels of TNF-α, IL-6, IFN-γ are observed in the blood, CSF and central nervous system (CNS) lesions of MS patients in active phase, while TGF-β and IL-10 are increased in remission or inactive phase.Therefore alteration in cytokine production may influence the susceptibility to MS and this alteration could be due to gene polymorphism. The aim of present study was to analyze whether there was an association between above cytokines' gene polymorphism and susceptibility to MS in the Iranian population.In this study, we screened genomic DNA samples from 98 clinically definite MS patients and 118 healthy controls, using polymerase chain reaction-sequence-specific primers (PCR-SSP). Cytokine gene polymorphisms were determined for TNF-α promoter-308 (G to A), IL-6 promoter-174 (G to C), IL-10 promoter-592 (C to A), -819 (C to T), -1082 (G to A), TGF-β codon 10 (C to A), codon 25 (G to C) and IFN-γ intron 1 + 874. The results indicated that MS patients significantly showed a higher TNF-α GG and TGF-β T/T G/G, T/C G/C genotypes compared to controls. However, our findings suggest that there could be genetically a dysregulated production of cytokines in Multiple Sclerosis and risk of the disease increase in low producers of TNF-α. PP10-05 A new developed culture method efficiently supports the growth of Chamydia pneumoniae in PBMC and CSF samples from patients with multiple sclerosis Chlamydia pneumoniae (Cpn), has been associated with various neurological disorders including multiple sclerosis (MS) on the basis of PCR findings. A question still open concerns the difficulty to cultivate and directly demonstrate Cpn in clinical specimens. Fresh CFS and PBMC samples were obtained from 8 patients with neurological and MRI abnormalities suggestive of MS. Specimens were inoculated on Hep-2 cells in duplicate wells, and incubated in CO 2 . A part of grown cells were investigated with immunofluorescence (IF) using Cpn-monoclonal antibodies. To increase Cpn inclusions, the resting wells were centrifuged and incubated with additional centrifugations on the 3rd and then on the 4th and 5th culture day, with medium refreshment at 72 h only. Culture supernatants underwent touchdown n-PCR assay targeting 16S rRNA, Momp and HsP 70 gene of Cpn. 3/8 patients were culture positive. CSF n-PCR was negative when DNA was directly extracted, whereas was positive in 1 (16S RNA) and 2 (MOMP) cultured samples when DNA was extracted from the 3rd day culture supernatants. PBMC n-PCR was positive for all genes, in higher positive rates than cultured CSF, when DNA was extracted from culture supernatants at 3 or 6 days. IF, performed before and after culture, was positive in only one PBMC sample. The results obtained confirm the efficacy of this culture system and suggest the presence and viability of Cpn. PP10-06 Serological profile of the immune response to microbes in identical twins discordant for multiple sclerosis Based on the nationwide series of Italian twins with multiple sclerosis, we performed a co-twin control study on 70 pairs in order to investigate causative factors for MS. Among 80 variables possibly associated with MS etiology, those positevely or negatively associated with predisposition and concordance in twins were mainly linked to infection.This finding prompted us to investigate quantitative serological profiles of immune response against microbes, that are putatively involved in MS etiology, in identical twins discordant for MS.Serum antibodies to EpsteinBarr (EBV) antigens, CMV, Varicella zoster (VZ), red measles, Herpes simplex 1, 2 and 6 viruses and Bordetella pertussis were quantified in 9 twin pairs discordant for disease 1 concordant pair and 1 control unaffected twin pair.The serum antibody titers were compared between affected and unaffected twins.Elevations in affected vs unaffected twins were significant for IgG anti-VCA (RU/ml, 1413.49 vs. 957, p < 0.05), a trend was found for anti-EBNA-1 and 2. No differences were found for the other microbes. Finally, the concordant twin pair and the unaffected twin pair showed comparable serological profiles.This finding supports the role of infections in the etiology of MS. The serological profiles of discordant twins showed differenced in the reactivity to VZV and EBV. The enhanced immune response to EBV in the affected twins is in keeping with previous work on sporadic patients. Reactive oxygen species (ROS) play a crucial role in several processes underlying the pathogenesis of multiple sclerosis (MS). They are involved in transendothelial monocyte migration, myelin phagocytosis and breakdown, oligodendrocyte damage and axonal degeneration. Exposure of cells to high levels of ROS induces oxidative stress, which leads to the activation of the nuclear transcription factor Nrf2. Activation of this transcription factor induces expression of antioxidant response elements (ARE)-regulated genes. These genes encode for endogenous enzymes that regulate cellular redox status and offer protection against oxidative stress and inflammation. Thus, expression of Nrf2/ARE proteins may act as a sensitive indicator of oxidative stress. Hence, we investigated the distribution of various endogenous antioxidant enzymes, including heme oxygenase, superoxide dismutase, peroxiredoxins, catalase and NAD(P)H:quinone oxidoreductase in different MS lesion types. We showed for the first time that antioxidant enzymes are highly upregulated in active demyelinated MS lesions, particularly in hypertrophic astrocytes and myelin-laden macrophages. We speculate that increased antioxidant enzyme expression may reflect an endogenous defense response and may compensate ROS-mediated cellular toxicity. Compounds that activate the Nrf2-ARE signalling pathway, via expression of detoxifying enzymes, may be a potential therapeutic target for future treatment strategies in MS. PP11-02 Reduced expression of MHC antigen-processing machinery molecules in margins of Multiple Sclerosis lesions: Clues for antiinflammatory countermechanisms at the lesion border? It is assumed that in Multiple Sclerosis (MS) lesions reactivation of infiltrating autoreactive lymphocytes is most likely mediated by local antigen presenting cells (APCs), such as microglia/macrophages. Antigen presentation requires major histocompatibility (MHC) antigen-processing, a complex interplay a several proteins belonging to the antigen-processing machinery (APM). In order to evaulate the regulation of APM molecules in relation to MS lesion formation, we investigated the expression of various MHC APM molecules in human brain specimens from MS patients (n =4) by means of immunohistochemistry. In relation to MHC class II, microglia/ macrophates exhibited 2 to 3-fold higher expression of MCH APM molecules in active (early-active, late-active and chronic-active) than in inactive (chronic-inactive) MS lesions. Interestingly, albeit higher frequencies of microglia and macrophages in borders of active lesions were observed, the expression of numerous MHC APM components such as delta, low molecular weight protein-2 (LMP-2), LMP-7, LMP-10, transporter associated with antigen zprocessing-1 (TAP1), calnexin and calreticulin was 1.5 to 2 fold higher in lesion centres than in lesion borders or normal appearing white matter (NAWM). In contrast to this, chronic-inactive MS lesions were characterized by comparable amounts of MHC APM expression in lesion borders and centres. Our data demonstrate microglia cells and macrophages to have the capacity to process and present antigens alike professional APCs. Reduced expression of MHC APM molecules in lesion borders in comparison to lesions centers could suggest antiinflammatory countermechanisms circumventing the broadening of MS-lesions. PP11-03 Both aquaporin-1 (AQP1) and aquaporin-4 (AQP4) are expressed in cultured human astrocytes and non-hypertrophic astrocytes in brains of multiple sclerosis Jun-ichi Satoh a , Hiroko Tabunoki a , Yusuke Nanri b , Takashi Yamamura b , Kunimasa Arima c and Hidehiko Konno d a Department of Bioinformatics, Meiji Pharmaceutical University; b Department of Immunology, NCNP; c Department of Neuropathology, NCNP, Tokyo; d Department of Neurology, Nishitaga National Hospital, Sendai, JapanObjective: To identify the expression of aquaporins (AQPs) in human neural cells. Background: AQPs constitute an evolutionarily conserved family of integral membrane water transport channel proteins. AQP4 is a target antigen for NMO-IgG in opticospinal multiple sclerosis (OSMS), although the mechanism underlying the highly selective lesion distribution in OSMS remains unknown. Methods: AQP1 and AQP4 expression was studied in cell cultures and brain tissues of four conventional MS cases by RT-PCR, Western blot and immunochemistry. Results: Human astrocytes in culture constitutively expressed both AQP1 and AQP4 on the cell surface. AQP4 protein levels were elevated in astrocytes by exposure to IFNG, but not by TNFA or IL-1B, while AQP1 was unaffected by any cytokines. Varying numbers of non-hypertrophic astrocytes with elaborated processes contacting blood vessels, but neither macrophages nor neurons, expressed intensely both AQP1 and AQP4 in MS and non-MS brains. Conclusions: AQP1 and AQP4 were expressed predominantly in nonhypertrophic astrocytes widely distributed in the whole CNS, suggesting a redundant role of both AQPs in astrocyte water balance, and an involvement of undefined regional factors in the selective lesion distribution in OSMS. PP11-04 Vα7.2-Jα33 invariant T cells accumulate in multiple sclerosis and CIDP lesions in contrast to a deficiency of Vα24-JαQ NKT cells Zsolt Illes a,b , Takashi Yamamura a a Department of Immunology, National Institute of Neuroscience, Tokyo, Japan and b Department of Neurology, University of Pecs, HungaryDeficiency of Vα24-JaQ NKT cells has been described in several autoimmune diseases, and we have observed absence of Vα24-JαQ NKT cells in multiple sclerosis (MS) lesions while conventional Vα24 T cells infiltrated MS plaques. This selective deficiency of invariant Vα24-JαQ NKT cells in MS prompted us to examine mucosal associated invariant T cells (MAIT) in nervous system autoimmunity, considering the proposed complementary functions, similarity and NK1.1 expression in the mouse homologues.Using RT-PCR SSCP method, expression of the reported four invariant TCR chains (Vα4, Vα7.2, Vα19 and Vα24) was examined in 25 MS lesions and 10 inflammatory demyelinating lesions of the peripheral nerves of chronic inflammatory demyelinating polyneuropathy (CIDP). Following RT-PCR with Vα and Cα primers, the amplicons were electrophoresed in non-denaturing SSCP gel. DNA was hybridized with biotinylated internal Cα-, Jα-specific or invariant junctional probes.Our data indicate that invariant Vα7.2-Jα33 MAIT cells infiltrate autoimmune lesions of both the central and peripheral nervous system in contrast to Vα24-JαQ NKT cells, which were only found in CIDP lesions. No other invariant T cells could be identified beside these two subsets in any lesions. In addition, presence of both or one of the Vα24 or Vα7.2 NKT cell subsets was associated with expression of IL-4 in autoimmune demyelinating lesions of the PNS, proposing their anti-inflammatory role. The differential expression of the two subsets in MS lesions suggests that MAIT cells may complement NKT cells and substitute deficiency of invariant Vα24-JαQ T cells in MS. PP11-05 Chemokine gradients play a role in lesional expansion of secondary progressive multiple sclerosis In secondary progressive (SP) multiple sclerosis (MS), there are slowly expanding demyelinating lesions with only modest inflammatory cell cuffing. Recently, evidence is accumulating that chemokines play an important role, not only in the leukocytes recruitment into the inflammatory site, but also in the recruitment of brain parenchymal cells. To elucidate the pathomechanisms of SPMS in terms of glial activation, we have investigated the expression of chemokines, MCP-1/CCL2 and IP-10/CXCL10, and their receptor, CCR2 and CXCR3 in the demyelinating plaques. Immunohistochemical analysis of autopsy brain tissues provided by UK Multiple Sclerosis Tissue Bank revealed that numerous hypertrophic astrocytes were observed at the rim, but not in the center, of the chronic active lesions. Microglia/macrophages phagocytosing myelin debris were also found at the lesion border. In contrast, T cell infiltration was minimal in these plaques. Characteristically, there were abundant immunoreactivities for MCP-1/ CCL2, IP-10/CXCL10, CCR2 and CXCR3 at the rim of the lesions with ongoing demyelination, whereas these immunoreactivities were relatively weak in the center of the lesions, forming chemokine gradients. Double immunofluorescense staining revealed that both chemokines and their receptors were expressed by hypertrophic astrocytes, while only chemokine receptors were expressed by MHC class II-positive microglia/macrophages. These findings suggest that chemokine expression by astrocytes plays an important role in microglia/macrophage activation and expansion of demyelinating lesions in SPMS. Targeting chemokines in SPMS could therefore be a powerful therapeutic approach to inhibit lesional expansion. PP11-06 Death receptors in the CNS: A role for decoys? Raine a a Albert Einstein College of Medicine., New York, USAThe mechanism by which myelin and oligodendrocytes are selectively targeted during lesion pathogenesis in multiple sclerosis (MS), is unknown but has been linked to tumor necrosis factor (TNF)-related damage and may be due to expression of TNF-related death-inducing surface receptors. The present study was undertaken to investigate by morphological and biochemical techniques, whether the death receptors DR3, DR6 and four receptors of TNF-related apoptosis-inducing ligand (TRAIL) -DR4, DR5, decoy receptor-1 (DcR1), and DcR2, were expressed in MS lesions and whether expression correlated with disease activity. The results showed that reactivity for DR molecules varied among microglia, astrocytes, and oligodendrocytes in all CNS tissue studied and on inflammatory cells in active MS lesions. DR expression was not specific to MS and was seen at different levels in other neurological diseases (OND), and normal CNS tissue. To examine the functional implications of DR expression, human oligodendrocytes grown in vitro, were exposed to TRAIL, and apoptosis measured by FACS using the TUNEL technique. The findings both in vivo and in vitro show that although oligodendrocytes in MS, OND and normal CNS express DR5 and display apoptosis when treated with TRAIL in vitro, there was negligible evidence of oligodendrocyte apoptosis in and around MS lesions. These findings suggest that the loss of oligodendrocytes in MS is probably the result of a non-apoptotic mechanism and that there is preservation around and within some lesions that may be linked to the presence of decoy receptors. PP11-07 Confocal immunofluorescence microscopy of optic neuritis John W. Rose 1,3 , Veda L. Tsoi 1 , Kenneth E. Hill 1,3 , Judith Warner 4,5 , and Noel G. Carlson [1] [2] [3] Objective: Optic neuritis (ON) is a demyelinating inflammatory disease of the optic nerve which may occur as an isolated disease or related to multiple sclerosis (MS). Methods: Pathologic optic nerves were obtained at autopsy from a patient with clinical recovery from clinically isolated ON. Normal optic nerves and ON tissues were probed with antibodies to pathologic antigens including myelin basic protein (MBP) fragment, the inducible form of nitric oxide synthase (iNOS), macrophage markers CD14 and CD64, nitrotyrosine, and cyclooxygenase (COX-2). We also examined myelin basic protein (MBP), the oligodendrocyte marker cyclic nucleotide phosphodiesterase (CNPase), and glial fibrillary acidic protein (GFAP). Results: In the affected pathologic nerve, iNOS + macrophages/microglia, iNOS + astrocytes, COX-2, and nitrotyrosine were observed. iNOS and COX-2 were occasionally observed in the unaffected nerve. Decreased expression of MBP and CNPase was seen in the pathologic optic nerves, along with evidence of gliosis and ongoing myelin degradation indicated by the presence of MBP fragment. Conclusions: The immunopathology of clinically isolated optic neuritis from this individual resembles that observed in active MS plaques. Despite clinical recovery, there was evidence of ongoing inflammation and demyelination. PP11-08 CD200-CD200 receptor interaction in the regulation of microglia and macrophages in multiple sclerosis Nathalie Koning 1,2 , R.M. Hoek 2 , I. Huitinga 1 1 Netherlands Institute for Neurosciences, Amsterdam, the Netherlands; 2 Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, the NetherlandsThe membrane glycoprotein CD200 (OX2) is highly expressed on neurons. In mice, CD200 has been shown to provide an inhibitory signal to cells of the myeloid lineage, including macrophages and microglia, via the CD200 receptor (CD200R) (Hoek RM et al. As activated macrophages and microglia are thought to be pivotal to the development of inflammatory lesions in the central nervous system of patients with MS, we hypothesize that reduced expression of CD200 is involved in activation of these normally quiescent cells. Therefore, we first study the expression of CD200 and CD200R in post-mortem frozen brain tissue from MS patients and controls by immunohistochemistry. Secondly, we use the laser dissection microscope to isolate well defined areas (rim and centre) of chronic active and chronic inactive white matter MS lesions. In these areas, gene expression profiles of CD200 and CD200R in relation to other molecules involved in myeloid cell regulation are determined by real time quantitative PCR, and compared with normal appearing white matter from both MS patients and healthy controls. Preliminary results show upregulation of CD200 in the rim of chronic active lesions, whereas in the rim and centre of chronic inactive lesions, CD200 is downregulated.Grant: Dutch foundation 'MS Research'. Brain tissue: Netherlands Brain Bank. PP11-10 Glutamate excitotoxicity damages demyelinated axons Glutamate excitotoxicity has recently been implicated in the lesion pathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). AMPA receptor antagonists have been shown to protect oligodendrocytes and axons in EAE, without altering the inflammatory response. Only oligodendrocytes, but not axons, are known to express AMPA receptors and axonal damage from excitotoxicity is believed to require the presence of oligodendrocytes [Underhill S.M., Soc. Since axons are lost in chronic demyelinated MS lesions, we examined the hypothesis that axons are vulnerable to glutamate excitotoxicity in the absence of oligodendrocytes. The cuprizone toxicity model was utilized in 4 week-old mice to eliminate oligodendrocytes and myelin from CNS white matter tracts. After 8 weeks of oral cuprizone administration, absence of myelin and oligodendrocytes in the corpus callosum was confirmed by Luxol fast blue and anti-CNPase staining. Microstereotactic infusion of the glutamate receptor agonist, AMPA, but not control vehicle, into the demyelinated corpus callosum resulted in substantial axonal damage as assessed by axon-specific Thy1-YFP fluorescent labeling. Extracellular glutamate is likely increased in chronic MS lesions due to excess glutamate production and/or decreased glutamate transporter expression. Excitotoxicity could therefore contribute to accumulation of axonal loss in MS lesions and may thus represent a target for neuroprotective intervention. PP11-11 Axonal sprouting in the adult CNS elicits long-term increases in the number of oligodendrocytes and length of myelinated fibres Remyelination is often incomplete or absent after demyelination in the CNS. Reasons for this attenuated repair might involve inhibitory factors within areas of myelin destruction, failure of demyelinated axons to support myelinating oligodendrocytes, or intrinsic limitations in the capacity of adult oligodendrocyte precursor cells. In the mature CNS, fibres with axonal segments lacking myelin sheaths can be generated experimentally by induction of axonal sprouting. We investigated the ability of axonal sprouting to induce a myelination response in the stratum radiatum and lucidum of the hippocampal CA3 region (srl-CA3) in the adult murine CNS, a region with pure sprouting and no degeneration. Using transection of the entorhino-hippocampal perforant pathway and an oligodendrocyte specific transgenic marker, we demonstrated that axonal sprouting in the srl-CA3 resulted in a significant 28% increase in the number of oligodendrocytes and a marked myelination response with a 41% increase in the length of myelinated fibres. Scattered proliferation of NG2 + oligodendrocyte precursor cells suggested a slow recruitment of myelinating cells. In contrast, in the dentate gyrus where degeneration occurs there was a strong NG2 + cell response but no subsequent change in oligodendrocyte numbers. Our study demonstrates that newly sprouted axons are able to elicit a myelination response in the mature CNS. PP11-12 Syncytin-1 mediates endoplasmic reticulum stress in a transgenic mouse model of multiple sclerosis Background: Syncytin-1 is a complex human retroelement envelope protein, which is up-regulated in the CNS of patients with MS and also contributes to oligodendrocyte death and demyelination although the underlying pathogenic mechanisms remain unknown. Methods: We developed a transgenic mouse line that selectively expressed Syncytin-1 in the CNS, together with performing analyses of host responses in brains of MS and non-MS patients and in cultured human neural cells. Results: Implantation of TNFα into the corpus callosum of Syncytin-1 transgenic mice caused neuroinflammation, neurobehavioral deficits and oligodendrocyte injury while also activating the endoplasmic reticulum (ER) stress-related genes, GADD153/CHOP, BiP, PERK, Grp58/ERp57, OASIS and iNOS, compared to wild type littermate controls. Similar findings of ER stress gene and iNOS induction were observed in the brains of MS patients. Syncytin-1 induced both OASIS and iNOS expression in astrocytes in conjunction with the repressive transcription factor, Egr1, which binds the promoter of ASCT1, a putative Syncytin-1 receptor. Astrocyte dysfunction resulting from ER stress was characterized by diminished levels of ASCT1 and trophic factors, particularly IGF-1. Discussion: The mechanism of oligodendrocyte damage and demyelination described herein represents a novel pathway by which over-expression of a human endogenous retroelement protein, Syncytin-1, leads to astrocyte dysfunction with adverse consequences in terms of oligodendrocyte viability and function. PP11-13 The RR-mouse: A double-transgenic SJL/J mouse model of spontaneous relapsing-remitting Multiple Sclerosis Bernadette Pöllinger 1 , Gurumoorthy Krishnamoorthy 1 , Hans Lassmann 2 , Hartmut Wekerle 1 , Andreas Holz 1 1 MPI Neurobiology, Martinsried, Germany; 2 Institute of Neurology, University of Vienna, AustriaWe first generated transgenic mice with a TCR specific for MOG peptide 92-106 in context of I-A s . The paired TCR genes (Vα8.3 and Vβ4) from an encephalitogenic T cell clone, were injected into fertilized oocytes of FVB origin. The transgenic founders were backcrossed into the SJL/J and B10.S strains.Seven transgenic lines expressed the transgenic TCR with low (<10%), medium (35%) and high (95%) frequency. All transgenic T cells responded vigorously to MOG in vitro, produced IFNγ and TNFα and transferred EAE upon activation.TCR MOG -high transgenic mice, line F1640, were crossed with IgH MOG knock-in mice (IgH MOG ) expressing the H chain of an anti-MOG antibody, 8.18C5.Double transgenic mice (SJL/JN3) spontaneously developed EAE in 100% of females and 60% of males within 3 months of age, while >20% of TCR MOG SJL/J mice and none of the IgH MOG mice developed spontaneous RR-EAE.In 89% of females, and 33% of males EAE took a relapsing-remitting course with an initial bout of ataxia and weight loss followed by complete recovery within few days, and, about 2 weeks later, a first relapse, either with ataxia (33%) or classical EAE (67%). Further relapses left neurological residua.The inflammatory lesions, dominated by macrophages and CD4 + T cells, faithfully reflected nature and timing of the clinical disease episodes by location and cellular composition of the lesions. PP11-14 Hypomyelination as a consequence of altered MBP gene transcription Stefanie Gaupp a , Glaucia Furtado b , Joseph Arezzo a , Sergio Lira b and Cedric S. Raine a a Albert Einstein College of Medicine, NY, USA; b Mt. Sinai Medical School, NY, USA Delayed or immature myelination has been noted in a number of transgenic models in which the transgene in question was under the control of a myelin-related promoter expressed by oligodendrocytes. During a study on mice overexpressing CCL2 (MCP-1) a chemokine involved in monocyte recruitment, in which the chemokine was under control of a myelin basic protein promoter and was produced by oligodendrocytes, we noticed that, widespread evidence of hypomyelination was apparent throughout the CNS in mice up to four months of age, most pronounced in optic nerves and subpial zones of spinal cord. In these areas, oligodendrocytes retained an immature phenotype, structurally and immunocytochemically, the vast majority (>75%) of axons were non-myelinated and of small caliber, the remainder thinly myelinated, and astrocytes and their processes invested the naked axons. Apart from an occasional monocyte within the CNS, breakdown of myelin and oligodendrocyte pathology were not prominent. In older animals, deeper white matter was more normally myelinated but interstitial parenchyma was intensely gliotic and there was some evidence of apoptosis of oligodendrocytes. Electrophysiology for visual evoked potentials showed responses to be delayed and substantially diminished in peak amplitude compared to age-matched, wild-type littermates. These findings underscore the need for the careful analysis of CNS architecture in models involving transgenes expressed by glial cells. Poster Session 12: MS-immunological studies We have shown that CD8 T-cells are the major population exhibiting clonal alterations of T-cell receptor CDR3 length distributions in the blood of multiple sclerosis (MS) patients. We aimed to characterize the epitopes recognized by CD8 T-cells that are derived from myelin antigens (MBP, PLP, MOG). The most frequent Class-I MHC in our patients were HLA-A2, -A3, -B27, -B7 and -B44. Peptides able to bind to these MHC were selected by sequence analysis of antigens, synthesised and tested for their affinity for the corresponding MHC. Among the 16 MS patients, 9 exhibited an IFNg response following peptidic stimulation. Among the 8 NI tested, 2 had a similar response. There was a significant increase in the frequency of responding cells in MS patients versus NI (p < 0.05). There was no public recognition of myelin peptides, since each patient had a private response pattern. Moreover, one patient showed serial positive responses at 4 time points for 3 peptides and responded to new peptides during the follow up. Three patients, tested positive in the first stimulation, were negative in the second, corresponding to a relapse. Collectively, we have characterized new autoreactive peptides. MS patients have a higher frequency of responding cells in blood than NI. Both NI and MS patients respond to various autoreactive peptides, these responses fluctuate in time and may depend on the disease state. PP12-02 Calpain activity correlates with cytokine production in patients with multiple sclerosis (MS) MK Guyton, S Imam, A Das, A Haque, WR Tyor, SK Ray, NL Banik Medical University of South Carolina, Charleston, SC, USA Multiple sclerosis (MS) is a devastating T cell-mediated auto-immune disease of the CNS, The l mechanisms responsible for Th1/Th2 cytokine dysregulation seen in MS are unclear; however, increased expression/ activation of calpain may play are role. Our previous studies indicate that increased calpain activity correlates with production of the Th1 cytokine interleukin (IL)-2 in activated peripheral blood mononuclear cells (PBMCs), we hypothesize that calpain expression/activity will correlate with Th1/Th2 cytokine dysregulation in activated PBMCs from relapsing/remitting MS patients and that treatment with a calpain inhibitor will alter cytokine production. In order to investigate a role of calpain in Th1/Th2 cytokine dysregulation, PBMCs from relapsing/remitting MS patients and age/sexmatched controls were pretreated for 1 h with the calpain inhibitor calpeptin (100 μM) or vehicle (DMSO). Cells were then activated for 24 h with anti-CD3 (10 μg/ml) + anti-CD28 (5 μg/ml) and harvested for Western blot analysis of calpain expression and activity. Supernatants were collected for determining levels of Th1 (IL-2) and Th2 (IL-4, IL-10) cytokines. Preliminary data indicate that calpain expression and activity were significantly (p ≤ 0.05) increased in both activated and unactivated PBMCs from remission and relapse patients versus controls. IL-2 production was significantly increased in relapse patients, while IL-4 and IL-10 levels were higher in remission patients. Interestingly, pretreating PBMCs with calpeptin resulted in a decrease in both Th1 and Th2 cytokine levels. These data suggest that calpain plays an important role in the production of cytokines in patients with MS. Dendritic cells (DC) are potent antigen-presenting cells and are critical for onset of the immune responses generates against infections. Bacteria and viruses have been implicated in autoimmune disease pathogenesis. Indeed, Multiple Sclerosis (MS) relapses are frequently associated with infection, in some instances bacterial. The hypothesis of this study was to establish whether DC from bacteria-infected MS patients modified autoreactive T cells patterns of activation, thus triggering disease exacerbation. CD1 + , CD11c + , CD80 + , CD86 + , CD14 − , HLA-DR bright DC were separated from peripheral blood during 11 exacerbations linked to bacterial infections and 11 relapses without infections. DC antigenpresenting capacity was assessed with MBP, MOG, GM1, and GM3 specific autologous T cell clones (TCCs). TCCs stimulated with DC from infected MS patients showed maximal proliferation, and induced the secretion of IL-12, IL-17, and IFN-γ at 10 to 30 times less concentration than after incubation with DC isolated from uninfected individuals. Interestingly, CD1-restricted GM1 and GM3 TCCs incubated with DC from infected MS patients secreted IFN-γ, and IL-12 even in the absence of exogenous antigens. These activation patterns correlate with increased in T cell survival. DC from infected MS patients secreted more IL-12 and IL-18, and showed higher expression of the myeloid differentiation factor 88, as well as molecules CD1, B7-1, B7-2, B7-DC, CD40, CD83 and CCR7, as compared to DC from uninfected individuals. Overall, these results are consistent with the concept that during bacterial infections, DC play a critical role in MS relapse induction. PP12-04 BDNF production by immune cells of patients with relapsing remitting multiple sclerosis is reduced and is not responsive to CD40 stimulation A. Karni, D Azulay Neuroimmunology Laboratory, Department of Neurology, Tel Aviv Sourasky Medical Center, Sackler's Medical School, Tel Aviv University, Tel Aviv, IsraelWe studied the production of BDNF and IFN-γ from PBMCs and the immune regulation of neuronal protection in 14 untreated patients with relapsing remitting MS (RR-MS) and 18 healthy controls (HC). Cells were incubated in several conditions: with monoclonal antibodies (mAb) against CD3, CD40 or CD154 or with IL-6 or TNF-α. BDNF and IFN-γ in the supernatants were measured by ELISA. Supernatants of the different conditioned PBMCs were added to neuroblastoma cell lines under starving condition in order to study their neuro-protective potential, as measured by neuronal cell count, neurites count or length. Cellular analysis of BDNF production was done by flow cytometry. PBMCs of RR-MS patients secreted lower BDNF (1044 ± 377 pg/ml) compared to HC (1360 ± 386 pg/ ml, p = 0.046). Monocytes was found to be the dominant BDNF producers (28.0%), compared to T cells (1.0%) and B cells (0.8%). Stimulation of PBMCs from HC with either anti CD3 mAb, anti CD40 mAb or LPS enhanced BDNF secretion (1602 ± 666 pg/ml, p = 0.002; 1483 ± 605 pg/ml, p = 0.01; 2940 ± 1052 pg/ml, p < 0.001, respectively). Only anti CD3 mAb stimulation enhanced IFN-γ secretion (2249 ± 699 pg/ml vs. 31-71 pg/ml in other conditions, p < 0.05). Supernatants of PBMCs that were cultured with anti CD40 or LPS induced protective effect on neuroblastoma cell lines, while supernatants of anti CD3 stimulated PBMCs induced cell death. In summary, Lower BDNF production by immune cells in RR-MS is related to dys-regulation of BDNF via CD40 and may contribute the neuronal damage of RR-MS. Activated γδ T cells lyse human oligodendrocytes in vitro but the exact mechanism of lysis is unknown. There are many ways in which γδ T cells could exert cytotoxicity and we have investigated the possibility that they might participate in antibody-dependent cell cytotoxicity (ADCC) via their expression of Fcγ receptor III (FcγRIII, CD16). To investigate if γδ T cells derived from MS patients were able to cause specific injury to target cells via ADCC, we established an in vitro experimental system using a humanized anti-CD20 monoclonal antibody rituximab (Rituxan®) as the bridging antibody and CD20 + B lymphoma cells lines as targets. Cytotoxicity was measured with multiple parameter flow cytometry. Our results show that, with Effector:Target ratio (E:T) at or above 4:1, the ADCC-type specific cytotoxicity of γδ T cells against the Raji cell line was significantly increased by the presence of rituximab (p < 0.02). Our findings suggest that γδ T cells, via Fc receptor binding, could lyse potential targets. Antibodies to myelin proteins are increased in both MS serum and spinal fluid, raising the possibility that if γδ T cells bind these antibodies, they could become effective targeted killers of oligodendrocytes. PP12-06 Human glial cells express NKG2D ligands that trigger immune cytotoxicity: Implications for multiple sclerosis Immune cells such as CD8, CD4 and gamma delta T cells are identified in lesions within the central nervous system (CNS) during inflammatory diseases such as multiple sclerosis (MS), suggesting that they are actively involved in the oligodendrocyte/neuronal injury. Human NK cells, CD8 + T cells and gamma delta T cells express NKG2D, a stimulating or costimulating receptor recognizing multiple ligands. NKG2D ligands, usually expressed on stressed/damaged cells are aberrantly expressed on targets during autoimmune inflammatory diseases such as celiac disease and rheumatoid arthritis and can contribute to enhance cytotoxic immune effectors locally. We sought to determine whether NKG2D ligands are expressed on human CNS cells. NKG2D ligands were detected on primary cultures of human adult oligodendrocytes and fetal astrocytes. Proinflammatory cytokines increased their expression on astrocytes, suggesting that under inflammatory conditions, such ligands could be upregulated. In vitro killing assays using activated human gamma delta T cells (or NK cells) as effectors demonstrated that both oligodendrocytes and astrocytes are susceptible to NKG2D-mediated injury since NKG2D blockade consistently reduced the extent of death. Immunohistochemical analyses suggest that NKG2D ligands are selectively detected in MS lesions but are absent from normal appearing white matter. These results imply that the recognition of NKG2D ligands on oligodendrocytes and astrocytes by NKG2D bearing immune effectors could contribute to cytotoxic responses especially in inflammatory conditions such as those occurring in MS. PP12-07 Reduced interferon-type I induced STAT4 activation and interferon type 1 receptor expression on peripheral blood mononuclear cells of interferon-naive relapsing remitting multiple sclerosis patients C.S. Constantinescu, A. Fahey, R.A. Robins University of Nottingham, Nottingham, United Kingdom Multiple sclerosis (MS) is an inflammatory demyelination disease of the central nervous system characterised clinically by neurological deficits which are often relapsing and remitting. Type 1 interferons (IFN), comprising IFN-beta and IFN-alpha, have immunomodulatory effects that can be beneficial in relapsing-remitting (RR) MS. Both IFN-beta and IFNalpha bind the same receptor and activate signalling pathways that include the phosphorylation STAT4 signal transduction molecule.The objective of this study was to compare the IFN activation of STAT4 in patients with MS who were not on treatment with IFN, and normal controls. We assessed STAT4 activation by intracellular staining with a phosphospecific antibody and flow cytometry.We investigated STAT4 activation by IFN-beta and alpha in 30 patients with RR MS and 20 age and sex matched healthy controls. IFN-beta is capable of inducing more STAT4 phosphorylation than IFN-alpha, but both were reduced compared to controls. To determine the mechanisms of reduced STAT4 activation in untreated MS, we quantified interferon type 1 receptor (IFNAR) expression on peripheral blood mononuclear cells of MS compared to controls, by flow cytometry using an antibody against IFNAR and by quantitative reverse transcriptasepolymerase chain reaction. Since we have shown that IFN-beta induces IFNAR, and MS patients have been shown to be IFN type 1deficient, this deficiency may be explained by reduced IFN-type 1 levels in MS. Current studies will determine whether treatment with IFN-beta enhances IFN responsiveness.Supported by MS Society of UK and Northern Ireland. PP12-08 Detection of NK-cells as putative regulators of multiple sclerosis disease activity during and after pregnancy Objective: Multiple sclerosis (MS) typically ameliorates during pregnancy but after the delivery there is an increase in the relapse rate. Our study was conducted as to better understand the immunoregulatory mechanisms behind this phenomenon. Methods: A prospective study including clinical, radiological and/or immunological follow-up of 42 MS-patients during pregnancy and six months into the postpartum period. Groups of healthy pregnant and nonpregnant persons and non-pregnant MS-patients were studied as controls. Laboratory investigations included measurement of intracytoplasmic cytokine production in peripheral blood lymphocytes and subtype analysis of T cells, B cells and NK cells both during and after pregnancy using immunofluorescence staining and FACS analysis. Results: Annualized relapse rate was significantly reduced during third trimester of pregnancy and increased after the delivery. Production of interferon-gamma by peripheral blood mononuclear cells was significantly less during pregnancy than in the postpartum period, resulting with an increased Th2:Th1 ratio during pregnancy in the MS-group but not in the control group.In MS, the Th2:Th1 ratio was 0.36 during pregnancy and 0.24 after the delivery. Diminished MS-disease activity during the last trimester of pregnancy was associated with an expansion of circulating CD56 Bright regulatory NK-cells.Simultaneously, the proportion of circulating CD56 dim NK-cells was reduced. Conclusions: CD56 Bright NK cells are known as efficient producers of the Th2 cytokine IL10 and hence, our observation of in vivo expansion of CD56 Bright NK cells during pregnancy may have implications for a potential role of CD56 Bright regulatory NK cells in the control of autoimmunity during pregnancy in MS. PP12-09 Differential modulation of the immune response in Multiple Sclerosis by viral and parasite infections J. Correale, M. Fiol, M. Farez, Institute for Neurological Research Dr. Raul Carrea (FLENI), Buenos Aires, Argentina Epidemiological and clinical observations suggest that viral infections may introduce a bias in immune responsiveness in Multiple Sclerosis (MS) patients, which in turn triggers disease exacerbations. Conversely, striking inverse correlation occurs between parasite infections and autoimmune diseases. The immune responses of 23 MS patients during viral infections, and 12 MS patients presenting parasite infections were studied. Numbers of IFN-γ, TNF-α, and IL-12 secreting cells, were higher in PBMC collected during exacerbations associated to viral infections, than during stable disease or exacerbations without infections. In parasite infected MS patients on the other hand, MBP-specific responses showed a significant increase in IL-10 and TGF-β, and a decrease in IL-12 and IFN-γ secreting cells, compared with uninfected MS individuals. In addition, only viral antigen (Ag) stimulation induced maximal myelin-Ag specific T cells effector response, at concentrations 20 to 30 times lower than native Ag alone. Moreover, MBP-specific T cell clones from parasite-infected patients were characterized by a cytokine profile similar to Th3 and Tr1 T cell subsets, and cloning frequency of CD4 + CD25 + FoxP3 + T cells was substantially increased in parasite infected MS patients compared both to uninfected and viral infected MS individuals. Overall, these observations suggest that whereas viral infections can increase PBMC response to myelin-Ag causing a Th1-like response increasing the relapse risk, parasite infections are able to induce regulatory T cells that on the contrary, can induce remission of the disease. PP12-10 Pregnancy as a natural modulator of disease activity in multiple sclerosis Disease activity in relapsing-remitting multiple sclerosis (MS) is significantly modulated during pregnancy, followed by a clear rebound activity after delivery. The biological mechanisms behind this phenomenon are largely unknown. Better insight in which natural immune and endocrine mechanisms are associated with clinically suppressed disease in MS can be of relevance for future management of the disease. In the Rotterdam Study on Pregnancy in MS, we aim to investigate the immune alterations that can be observed in the peripheral blood. We here investigated gene expression in the different stages before, during and after pregnancy. Blood monocytes were purified using MACS, RNA was extracted and gene expression was probed using Affymetrix RNA-arrays. Samples were assessed in longitudinal series of 4 time points per patient. After correction for multiple testing (22,000 gene transcripts), in a pilot study more than 100 genes were differentially expressed in the monocyte fraction during pregnancy compared with the non-pregnancy timepoint. The majority of genes was downregulated and most were linked to immune functions, such as chemokine family members and proinflammatory cytokines. This lends support to the hypothesis that during pregnancy also innate responses are attenuated. Moreover data will be correlated to serum Il-10, IL-17 and IL-23 levels (measured by ELISA), as well as clinical disease activity, such as exacerbation after delivery. PP12-11 Functional deficit in CD4 + CD25 + Foxp3 + regulatory T cells in multiple sclerosis Multiple sclerosis is believed to be a T cell-mediated autoimmune disorder of the central nervous system and may involve impaired immune regulatory mechanism. In this study, we examined the CD4 + CD25 + T cells isolated from the peripheral blood of MS patients and healthy individuals. We found that the cell number of CD4 + CD25 + T cells in MS was slightly higher than in healthy individuals, but the Foxp3 (a Treg specific gene) expression in CD4 + CD25 + T cells derived from MS patients was significantly lower than that from healthy controls. Furthermore, CD4 + CD25 + T cells isolated from MS patients had less inhibitory effect on T cell activation as compared to CD4 + CD25 + T cells derived from healthy controls. Our results suggest that CD4 + CD25 + T cells in MS represent mostly in vivo activated inflammatory T cells that lack the expression of Foxp3 and inhibitory function. The functional deficits of CD4 + CD25 + Foxp3 + regulatory T cells might contribute to hyperactivity of T cells of pro-inflammatory potential in MS. PP12-12 Mobilization of CD34 + myeloid progenitors in the blood of multiple sclerosis patients Recent data indicate that blood monocytes form a heterogeneous cell population, which comprises a subset of progenitor cells expressing CD34, a surface molecule found on haematopoietic progenitors, haematopoietic stem cells and microglia. These CD14 + /CD34 + cells are endowed with a high proliferative potential and display differentiation potential toward macrophages, dendritic cells and possibly microglia. Here, we assessed the behavior of blood-circulating CD34 + myeloid cells in multiple sclerosis (MS) patients. We analyzed by FACS the peripheral blood mononuclear cells (PBMCs) obtained from 10 relapsing-remitting MS (RRMS), 10 primary progressive MS (PPMS) and 10 healthy control subjects. In parallel experiments, we performed a functional assay by culturing PBMC in methylcellulose supplemented with macrophagecolony stimulating factor. The percentage of CD14 + cells, MHC class II + cells or CD86 + cells was unchanged in MS patients as compared to controls. In contrast, both RRMS patients and RPMS patients showed increased percentages of CD34 + myeloid cells (4.59 ± 0.5% in RRMS patients, 4.57 ± 0.52% in RPMS patients, 2.71 ± 0.23% in controls, ⁎⁎⁎ p < 0.005, Student's t test). Such an increase was partly due to the specific expansion of CD14 + /CD34 + cells as shown by double-staining experiments. Accordingly, we could generate large colonies of macrophages (at least 50 cells per colony) in MS patients (2 out of 5) but not controls (0 out of 5). The migratory behavior and fate of such cells under neuro-inflammatory conditions is currently being assessed in mice. PP12-13 Secondary progressive in contrast to relapsing-remitting multiple sclerosis patients show a normal CD4 + CD25 + regulatory T cell function and FOXP3 expression In the present study, the phenotypic and functional characteristics of CD4 + CD25 + regulatory T cells (Tregs) isolated from the peripheral blood of patients with relapsing-remitting (RR, n = 31) and secondary progressive (SP, n = 21) multiple sclerosis (MS) were investigated. No significant quantitative abnormalities in CD4 + CD25 + T cells from RRand SP-MS patients were detected. However, the mean suppressive capacity of Tregs towards anti-CD3 induced responder cell proliferation was found to be significantly lower in RR-MS patients (43 ± 7%) as compared to Tregs from SP-MS patients (80 ± 3%). The suppression of MS derived Tregs is correlated with disease duration but not with age indicating that Treg function is more affected in the early phase of the disease process. Tregs from SP-MS patients showed normal levels of FOXP3 mRNA in contrast to Tregs from RR-MS patients that had a reduced FOXP3 expression.Recently, we developed a CFSE based assay to compare myelin specific responses of CD4 + T cells and Treg depleted CD4 + T cells (i.e. CD4 + CD25 − T cells). CD4 + T cell responses against myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) were increased in Treg depleted CD4 + T cells of healthy controls. We are currently using this indirect approach to analyze Treg suppression of myelin CD4 + T cell reactivity in MS patients.Taken together, these data are the first to demonstrate differences in function and FOXP3 expression between CD4 + CD25 + T cells from patients with RR-and SP-MS. In addition, Tregs appear to control myelin specific T cell responses in healthy subjects. PP12-14 Enhanced levels of anti-myelin antibodies in multiple sclerosis patients as assessed by flow cytometry Antibodies are thought to contribute to CNS inflammation and demyelination in a subgroup of multiple sclerosis (MS) patients. Antibodies directed against a number of myelin proteins can be measured in serum of multiple sclerosis (MS) patients, but no single antigen has been associated exclusively with MS. Studies in animal models suggest that antibodies with demyelinating capacity are directed against native conformational and posttranslationally modified epitopes. The current methods to detect anti-myelin antibodies often do not allow recognition of such epitopes, because recombinant or denatured proteins are used as antigens.We have developed a reproducible, flow cytometry-based assay to detect serum antibodies that bind to human whole myelin, allowing detection of antibody binding to conformational and post-translationally modified epitopes.Using this assay, anti-myelin antibodies were measured in serum of 152 MS patients and 40 healthy donors (HD). At the group level, anti-myelin IgM levels in MS were slightly higher than in HD (p = 0.012, Mann-Whitney). Enhanced anti-myelin IgM or IgG levels were found in all clinical subgroups of MS patients (RR, SP and PP), and the anti-myelin antibody levels did not correlate with disease severity (EDSS, MFSC).In summary, the myelin flow cytometry assay detects increased levels of anti-myelin antibodies in a subpopulation of MS, indicating this assay may provide a valuable biomarker to identify patients with antibody-mediated inflammation. PP12-15 Proteomic analisys of autoantibody reactivity to neural antigens in sera and cerebrospinal fluid of multiple sclerosis patients Several proteins of the central nervous system (CNS) have been investigated as potential targets for antibody-dependent immune response in multiple sclerosis (MS). In the present study we overcome such restrictive approach using a large panel of native protein antigens derived from white matter homogenate of human healthy CNS.We compared IgG repertoires in sera and cerebrospinal fluid (CSF) from 17 control subjects and 16 MS patients by immunoblotting after 2D-PAGE resolution.Results: The analysis of seric and CSF IgG reactivity after 2Dimmunoblotting showed altogether 349 spots. Among these, 117 spots were specifically recognized by MS patients; in particular, 73 were identified by seric IgG and 44 by CSF IgG. The majority of these spots was not recognized by corresponding sera, thus indicating a compartimentalized autoimmune response. Noteworthy, 7 spots detected by seric IgG and 7 by CSF IgG were present in >50% of MS cases and absent in control sera.Conclusions: The 2D-PAGE technique allowed us to increase the sensitivity of analysis compared to 1D-PAGE. The study of autoantibody reactivity against CNS antigens with this technique enabled the identification of about 117 spots in the serum and CSF of MS patients, which were not detectable in controls. Their characterization by mass spectrometry will provide important information regarding the target antigens involved in the autoimmune response in each MS subgroup. PP12-16 Autoreactive B-cell mediated immune response as an early fingerprint of neurological disorders associated with autoimmune processes To identify antigenic targets in autoimmune demyelinating disorders, we have used analysis of serum self-IgG responses associated with a proteomic approach (SERPA) in different clinical and experimental models of neurological disorders associated with autoimmune responses. In a first step, a global analysis, by Western blotting of serum self-IgG responses against healthy and multiple sclerosis (MS) human brain protein antigens allowed to discriminate patterns obtained in MS patients, healthy subjects and in the three clinical forms of MS. A cluster of discriminant antigenic bands were also found by studying sequential changes during the progression of Experimental Autoimmune Encephalomyelitis (EAE), manipulated either by anti-inflammatory drugs or by the enhancement or the depletion of CD4 + CD25 + regulatory T cells.To test the significance of such changes as potent hallmark of disease activity, serum self-IgG response was also evaluated in neuropsychiatric systemic lupus erythematosus (NPSLE) and in clinically isolated syndrome patients (CIS), the earliest clinical event in MS. Singular self-IgG patterns were found in NPSLE and prospective studies have shown that a significant number of patients with CIS present autoreactive pattern predictive of the development of clinically definite MS.Subsequently, a proteomic approach, was performed to characterize molecular targets involved. As previously noted in other organ specific autoimmune diseases, ubiquitous proteins appear as major discriminant targets found in MS, EAE and NPSLE. Only in some case, specific brain derived antigenic targets were found. Thus, SERPA provides a promising approach for investigating self candidate antigens involved either in regulatory or in pathogenic processes of autoimmune disorders and for the development of new diagnostic tools. PP12-17 Antigen microarrays identify unique immune repertoires in multiple sclerosis (MS) We used antigen microarrays to study the repertoire of serum antibodies in relapsing-remitting (RR), secondary progressive (SP) and primary progressive (PP) MS. Antigen microarrays consisted of 420 antigens including CNS-related autoantigens (e.g., MBP, MOG, PLP), lipids (gangliosides, galactocerebrosides) other autoantigens (e.g., insulin, GAD, collagen, GBM) and viral pathogens (e.g. EBV, herpes, varicella). To investigate natural autoantibodies, serum at a 1:10 dilution was assayed on two independent arrays and results analyzed using GeneSpring software. We found that we could distinguish RRMS, SPMS or PPMS from healthy controls by unique antibody patterns, but not by a single antibody reactivity. These antibody patterns were used to analyze the test set and correctly identified 75% of RRMS, 80% of SPMS and 94% of PPMS. The patterns we observed were different from those we found for other neurologic diseases. The discriminating patterns of antibody reactivity demonstrated that IgG was more informative that IgM, however, detailed analysis of IgG1 and IgG4 antibodies did not improve sensitivity. The discriminating antibodies we identified primarily targeted CNSspecific antigens, but also contained antibodies directed against the 60 kDa and the 70 kDa heat shock proteins (HSP60 and HSP70). IgM antibodies to EBV were associated to RRMS and SPMS. Our results demonstrate unique immune repertoires in MS that could be used as biomarkers for the diagnosis and evaluation of MS patients, and potentially to identify healthy individuals at risk for MS. PP12-18 What is the functional relevance of autoantibodies against myelin in people with multiple sclerosis? Many studies have identified antibodies against myelin and axonal proteins and glycolipids in people with multiple sclerosis (MS). In our studies, we have found increased levels of antibodies specific for myelin proteolipid protein (PLP) epitopes in sera of people with MS, compared to healthy controls and patients with other neurological diseases (OND). It is possible that the antibodies identified may not be particularly relevant to the disease process, as they may not recognize the antigen in its native configuration, which is likely to be necessary for the antibodies to be of functional relevance in MS.We have therefore been studying whether antibodies from patients with MS can recognize antigen in its native conformation, using human myelin opsonization assays and flow cytometry of stable cell lines expressing human myelin proteins. We find that antibodies from people with MS are significantly better at opsonizing myelin than antibodies from healthy controls or patients with OND. For the flow cytometry assays, stable cell lines have been generated expressing either, PLP and its alternatively spliced isoform, DM20, myelin basic protein (MBP) or oligodendrocyte-specific protein (OSP) attached to a fluorescent marker. The cells are incubated with serum samples from people with MS and controls, a FITC-labelled anti-human antibody is added, and the cells are read by a flow cytometer.This study will show whether the myelin-specific antibodies found in people with MS are likely to be of functional relevance in the disease. PP12-19 Antibodies produced by clonally expanded plasma cells in the cerebrospinal fluid of MS patients display CNS autoreactivity Oligoclonal IgG, the product of clonally expanded B/plasma cells, are a diagnostic hallmark in the CSF of MS patients. While increasing evidence supports a role of autoantibodies in the pathogenesis of demyelinating MS lesions, the antigenic specificities and pathogenic relevance of oligoclonal CSF IgG remain largely unknown. We performed single cell RT-PCR of expressed immunoglobulin genes on individual CD138 + FACS-sorted CSF-plasma cells from MS patients. Following sequence analysis we expressed paired heavy (H) and light (L) chain genes from clonally expanded plasma cells as whole recombinant human IgG1 in a eukaryotic expression system. Recombinant human monoclonal IgG1 were tested by Western blotting and immunofluorescence staining of brain tissue for their reactivity with CNS antigens. H chain variable region (IGHV) genes of expanded plasma cell clones from the CSF of 4 MS patients mostly utilize IGHV1, 3 and 4 subfamily genes. Some monoclonal recombinant antibodies derived from different patients showed specific reactivity with antigens present in brain tissue. In summary, we find signs of an antigen driven, CNSautoreactive humoral immune response in the CNS compartment of patients with MS. It remains unclear whether this autoantibody response is part of a primary immune response leading to or of a secondary immune response resulting from tissue destruction in MS. The pathogenic relevance of such antibody responses is subject to further investigations. PP12-20 Diagnostic value of CSF autoantibodies against heterogeneous nuclear ribonucleoprotein A1 and A2/B1 in multiple sclerosis patients Objective: The aim of this study is to draw a conclusion on the implication of anti-heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and/or A2/B1 antibodies (Abs) in multiple sclerosis (MS) and human Tcell lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Methods: Using recombinant hnRNP A1 and B1 proteins, Western blotting was conducted for detection of anti-hnRNP A1 and A2/B1 Abs in 168 CSF samples in fully masked conditions. Results: All enrolled cases had neurological abnormalities and were tested for CSF anti-HTLV-I Abs. Among the 102 cases with negative anti-HTLV-I Abs, the incidence of CSF anti-hnRNP A2/B1 Abs was the highest in MS (88.0%, n = 25), that was significantly different from the incidence in neurodegenerative diseases (15.5%, n = 20) (p < 0.0001), meningoencephalitis (38.1%, n = 21) (p = 0.0013), and inflammatory polyneuropathies (36.8%, n = 19) (p = 0.0013). Among the 66 cases with positive anti-HTLV-I Abs, the incidence of CSF anti-hnRNP A1 Abs were far lower in HAM/TSP (35.0%, n = 40) than in the group of other diseases (57.7%, n = 26). Interpretation: The present study confirmed our previous finding regarding the significant association of CSF anti-hnRNP A2/B1 Abs with MS but it failed to demonstrate any diagnostic value of CSF anti-hnRNP A1 Abs in HAM/ TSP. CSF anti-hnRNP A2/B1 Abs may provide a clue in the diagnosis of MS. [Objective] Helicobacter pylori (HP) infection has been previously reported to be related to various diseases. Neutrophil activating protein (HP-NAP) is capable of activating neutrophils and monocytes. The aim of this study was to disclose the prevalence of HP infection and the immune response to HP proinflammatory protein, HP-NAP, in Japanese patients with multiple sclerosis (MS). [Methods] We investigated the presence of anti-HP and anti-HP-NAP reactivity in serum of MS patients (n = 105) and compared them with healthy controls (n = 85). MS patients were clinically classified into two subtypes: opticospinal type (OSMS) or conventional type (CMS). [Results] We found that frequency of HP seropositivity was significantly lower in CMS patients (22.6%), as compared with OSMS patients and healthy controls (p = 0.0019 and p = 0.018, respectively). When analyzed separately by year of birth, for patients born after 1950, HP seropositive rate was significantly lower in CMS (20%) than OSMS patients (40.6%, p = 0.0483). Severe visual impairment (FS 5 or higher than 5) was significantly more common in HP-seropositive patients than seronegative patients in total MS patients (p < 0.05). EDSS score at final follow-up did not differ significantly between HP seropositive and seronegative groups. Anti-HP-NAP antibodies were found to be positive in 4 patients with MS and all of them were OSMS patients (8.2% positive). [Conclusions] Our study indicates a difference in HP seropositivity and the antibody to its proinflammatory protein, HP-NAP, between OSMS and CMS in Japanese. Although further studies are required, these observations may suggest distinction in the childhood environment in sufferers of these conditions. PP12-22 Role of chronic infection in the aetiology/pathogenesis of idiopathic parkinsonism Objective: to elucidate causality by systematic exploration of clinical clues, calling on wide-ranging laboratory/clinical expertise. Methods: Simple and reliable objective measurements of facets of parkinsonism underpin statistical modelling, hypothesis generation and efficacy studies. Results: To date, proof-of-principle that infection contributes to idiopathic parkinsonism (IP) has been provided by case studies and interim protocol analyses of an efficacy study contrasting effect, on the time-course of IP facets, of successful Helicobacter pylori (Hp) eradication vs. placebo, and vs. failure (Helicobacter 2005; 10:267-297) . 'Malignant' IP appears converted to 'benign', but marked deterioration accompanied failure. Categorisation, according to presence/absence of overt Helicobacter infection, was a useful therapeutic tool even in late IP with cachexia. Although Hp infection is overt in a minority of probands, the predicted probability of having the parkinsonian label depends on the serum immunoblot Hp antibody profile, with clinically-relevant gradients between this "discriminant index" and disease-burden and -progression. Indeed, as we begin to explore urea-breath-test negative IP, we are finding molecular evidence for infection in gastric biopsies, where there is no growth on routine culture (presumably due to low organism density). Moreover, early experience of open intervention suggests that it might prove as beneficial as in culture-positive cases. With eradication-failure, bolus-release of antigen from killed bacteria, could aggravate an effect of ongoing infection. The apparent importance of Helicobacter in the aetiology/pathogenesis of IP is not confined to those with overt infection. PP12-23 Anti-Nogo receptor autoantibody in the serum of multiple sclerosis Objectives: A myelin-associated neurite outgrowth inhibitor Nogo-A plays a key role in inhibition of axonal regeneration. Axonal damage beginning at the early stage of MS is responsible for permanent neurological deficits, although its molecular mechanism remains unknown. The aim is to study the prevalence of autoantibodies against Nogo-A and Nogo receptor (NgR) in the serum of MS. Methods: The antibodies were identified in the serum of 30 MS patients, 22 patients with non-MS neurological diseases (OND), and 22 healthy control (HC) subjects by Western blot using recombinant human Nogo-A-specific segment (NAS), the shared segment of Nogo-A and -B (NAB), Nogo-66 (N66), the non-glycosylated form of NgR, the glycosylated NgR (NgR-Fc), and myelin oligodendrocyte glycoprotein (MOG). Furthermore, 60% of MS, 18% of OND and 14% of HC showed anti-NgR-Fc IgG. Conclusions: Because IgG autoantibodies against N66, NgR and MOG are often detected in the serum of MS and controls, they do not serve as a MSspecific marker. We have measured cerebrospinal fluid (CSF) and serum levels of active matrix metalloproteinase-2 (MMP-2) and its tissue inhibitor TIMP-2 by Activity Assay System and ELISA, respectively, in 51 relapsingremitting (RR) multiple sclerosis (MS) patients, grouped according to clinical and Magnetic Resonance Imaging (MRI) evidence of disease activity, in 50 patients with other inflammatory neurological disorders (OIND) and in 50 patients with non-inflammatory neurological disorders (NIND). Statistical analysis was performed by Mann-Whitney, with Bonferroni post-hoc correction, and Spearman rank correlation coefficient tests. CSF and serum active MMP-2/TIMP-2 ratios were higher in MS than in NIND (p < 0.05 and p < 0.001, respectively) and in MRI inactive than in MRI active MS patients (p < 0.01 and p < 0.02, respectively) whereas CSF active MMP-2/TIMP-2 ratio was more elevated in MS than in OIND (p < 0.05). An intrathecal synthesis of active MMP-2 and TIMP-2 was greater in MS than in OIND and NIND (p < 0.001) and in MRI inactive than in MRI active MS patients (p < 0.05). In addition, a positive correlation (R = 0.303; p < 0.01) was found between serum active MMP-2/ TIMP-2 ratio and MS disease duration. Overall, these results suggest that a shift in MMP-2/TIMP-2 balance towards proteolytic activity of MMP-2 could be relevant in tissue repair of a subset of MS patients with MRI inactive disease and seem to indicate that serum active MMP-2/TIMP-2 ratio may represent a potential surrogate biomarker for monitoring MS disease duration. PP13-03 Cell surface adhesion molecules and cytokine profiles in blood and CSF in primary progressive multiple sclerosis Objective: We evaluated whether the expressions of adhesion molecules (AMs) and cytokines in blood and CSF could be used as markers of disease activity in primary progressive multiple sclerosis (PPMS). Methods: The expressions of AMs and the levels of 17 cytokines in patients with PPMS were compared with those in SPMS and controls and correlated with the volumes of focal and atrophic changes in brain magnetic resonance imaging (MRI). Results: The expressions of very late activation antigen 4 (VLA-4), lymphocyte function associated antigen 1 (LFA-1) and intercellular adhesion molecule1 (ICAM-1) in blood and CSF were higher in PPMS than in controls. Comparison between PPMS and SPMS showed higher levels of ICAM-1 in blood and CSF in PPMS, while the level of vascular adhesion molecule (VCAM-1) was higher only in blood. There was no difference in the levels of cytokines in serum or CSF between PPMS and SPMS or controls, but the evidence for intrathecal synthesis of IL-8 and MCP-1 was found in PPMS. The expressions of CSF VLA-4 in PPMS correlated with the total volume of cerebral lesions and the number of diffuse brain lesions in MRI, while the amount of LFA-1 in CSF correlated with the number of spinal T2 lesions. Conclusions: The upregulated expressions of AMs in blood and CSF and evidence for intrathecal synthesis of MCP-1 and IL-8 in PPMS indicate the importance of inflammatory changes in the pathogenesis of PPMS and suggest the use of these molecules as markers of disease activity in this MS subtype. PP13-04 Regulation of monocyte CD163 expression in multiple sclerosis used to treat relapses in multiple sclerosis (MS), but the response to this treatment differs among patients, suggesting differences in sensitivity to GC. Due to the induction of CD163 by GC, we wondered if CD163 might be valuable as a marker to predict glucocorticoid responsiveness. Finally, the in vitro inducibility of CD163 by GC correlated positively with the clinical response to GC, suggesting CD163 as a possible prognostic marker for GC sensitivity. PP13-05 Levels of serum IgM antibody against refolded recombinant human MOG predict responsiveness to corticosteroids for treatment of acute multiple sclerosis relapses Background: Anti-Myelin-Oligodendrocyte-Glycoprotein (MOG) antibodies (Abs) are discussed as biological marker in multiple sclerosis (MS) patients. therapy has been established for treatment of acute relapses in MS-patients. However, only scant information on the impact of corticosteroid treatment on (auto-) antibodies is available. Objectives: To establish an ELISA test system for detection of serum IgG and IgM Abs directed against refolded recombinant human MOG. To prospectively investigate whether acute relapses and subsequent HDMP therapy have an impact on serum anti-MOG Abs in MS-patients and whether anti-MOG Ab levels might predict the responsiveness to HDMP therapy. 25 MSpatients were then prospectively enrolled and serum samples were analyzed in remission before relapse, during acute relapse before HDMP treatment and 6 weeks after HDMP therapy. Results: Anti-MOG IgG (sensitivity 27.7% specificity 89.9%, p < 0.001) and anti-MOG IgM (sensitivity 23.4%, specificity 89.9%, p < 0.001) antibody levels were significantly increased in MS-patients compared to HC. Conclusion: The findings from this prospective study could be important for future decisions in patients with acute relapses, especially decisions regarding either dosing/duration of HDMP or escalating therapies. We demonstrated that in Multiple Sclerosis (MS) deregulation of programmed cell death (PCD) play key role in inducing or maintaining autoreactive immune phenomenon leading to demyelinating lesions.In the present study we investigated the PCD of myelin basic protein (MBP)-specific T lymphocytes in 47 relapsing-remitting (RR) MS patients with 29 acute (AMS) or 18 stable MS (SMS) and 30 Healthy Controls (HC).We analyzed by flow cytometry CD4 + CD8 + apoptotic and CD3 + proliferating cell percentage, by RT PCR the expression of different anti (FLIP, XIAP, and pro (BID, APAF-1) apoptotic genes in sorted CD4 + and CD8 + T cells stimulated with MBP peptides. Differences were analyzed by t-Student and Mann-Whitney tests. Percentage of apoptotic MBP specific CD4 + and CD8 + T cells decreased in AMS compared to SMS (p < 0.05) and HC (p < 0.05), higher proliferation index of MBP specific T cells was found in AMS and HC compared to SMS (p < 0.05). Higher expression, even thought no statistically significant, of anti and pro apoptotic genes was shown in RRMS compared to HC, however significant increase of anti apoptotic genes FLIP XIAP and Bcl-2, was observed in MBP-specific sorted CD8+cell of AMS compared to HC.The data obtained evidence specific activation of immune system against MBP in patients with AMS and in HC, but the increase of PCD in HC switch off the MBP specific T cell immune response, while, in AMS the decrease of apoptotic MBP specific T cell seems to be involved in the immune mediated destruction of myelin sheath. PP13-07 Differences in TRADD expression indicated by array analysis discriminates between MS disease subtypes Relapsing-remitting (RR) and primary progressive (PP) MS differ in their clinical course and most likely also their underlying pathomechanisms. This study aimed to identify biomarkers in association with subtypes of MS, using cDNA array analysis of blood mononuclear cells (PBMC) as an initial approach. Array analysis was performed using pooled RNA derived from PBMC of patients with stable RRMS or PPMS not on immunomodulatory treatment. Using real-time RT-PCR differential expression of candidate genes was quantified in individual MS patients of two independent patient cohorts containing both subgroups (a) 9 RRMS/9 PPMS and (b) 27 RRMS/42 PPMS.Gene expression array analysis identified 51 genes differentially upregulated in RR-MS and 11 in PP-MS. Out of several candidate genes re-analysed individually by quantitative RT-PCR the death domain containing protein TRADD involved in tumor necrosis factor receptor (TNFR) superfamily signalling was consistently expressed at higher levels in PP-MS patients compared to RR-MS and healthy controls. Results of Western blots and three-coloured FACS analysis for TRADD in different PBMC populations will be presented.Array analysis is a useful screening approach in pooled MS patient samples to identify differentially expressed genes. TRADD, a member of TNFR superfamily, is one of these candidate genes and may serve as biomarker to discriminate PPMS from RRMS patients. PP13-08 Inverse association between CSF levels of soluble HLA-G and Fas molecules in MS patients with no evidence of MRI disease activity We have studied by ELISA technique cerebrospinal fluid (CSF) levels of classical soluble HLA-I (sHLA-I), non-classical soluble HLA-G (sHLA-G) and soluble Fas (sFas) molecules in 65 relapsing-remitting (RR) multiple sclerosis (MS) patients, categorized according to clinical and Magnetic Resonance Imaging (MRI) evidence of disease activity, in 64 patients with other inflammatory neurological disorders (OIND) and in 64 patients with non-inflammatory neurological disorders (NIND). Statistical analysis was performed by Mann-Whitney, with Bonferroni post-hoc correction, and Spearman rank correlation coefficient tests. CSF levels of sHLA-G were higher in MS than in OIND and NIND (p < 0.001 and p < 0.01, respectively) and in MRI inactive than in MRI active MS patients (p < 0.01). CSF sHLA-I concentrations were more elevated in MS than in NIND (p < 0.02), in OIND than in NIND (p < 0.02) and in MRI active than in MRI inactive MS patients (p < 0.01). CSF levels of sFas were lower in MS than in OIND and NIND (p < 0.001 and p < 0.02, respectively) and in MRI inactive than in MRI active MS patients (p < 0.02). PP13-09 In vivo macrophage activity imaging in inflammatory brain lesions of multiple sclerosis using MRI Mitsunori KANAGAKI, Vincent DOUSSET, Bruno BROCHET, and Klaus G. PETRY University Victor Segalen Bordeaux 2, EA2966 Neurobiology of Myelin Disorders, Bordeaux, France Objective: In multiple sclerosis (MS), activated microglia and blood-borne macrophages play a pivotal role in the inflammatory process causing myelin and axonal loss. We present a new MR imaging modality to monitor in vivo macrophage brain infiltration in MS and its rat model of experimental autoimmune encephalomyelitis (EAE) with ultra-small superparamagnetic iron oxide (USPIO) nanoparticles. Methods: USPIO were intravenously given in both EAE rats and 10 MS patients during the acute attack. MRI-USPIO was performed 24 h later on MR scanners (1.5 T for humans, 4.7 T for rats) in comparison with gadolinium (Gd), MRI marker of blood-brain barrier permeability. In EAE rats, we evaluated histologically the underlying physiopathology with immunomarkers.Results: In acute EAE, USPIO enhanced lesions were detected before Gd enhancement. USPIO were proven to be localized in tissue macrophages. Two patients presented 2 lesions only enhanced by USPIO (negative for Gd), and the remaining lesions were detected by Gd only (Am J Neuroradiol, in press). In acute and relapsing EAE, MRI-USPIO allowed to monitor the efficacy of immunomodulatory therapies (Mult Scler 2004) and to predict, already at clinical EAE onset, the histopathological and handicap severity (Neuroimage, in press). MRI-USPIO is a promising tool to monitor in vivo the dynamic macrophage activity and its modulation upon therapeutics. PP13-10 Morphometry of the corpus callosum: An early marker of brain atrophy in patients with probable multiple sclerosis Shmuel Miron, Or Reshef, Yael Nissan, Anat Achiron Multiple Sclerosis, Sheba Medical Center, Ramat-Gan, Israel Background: Modern neuroimaging techniques provide information about brain structure. In multiple sclerosis (MS) it is possible to study axonal loss known to occur in the early stage of the disease. Irreversible axonal loss is associated with brain atrophy, and overtime lead to clinical disability. Objective: Investigate whether changes in the area of the corpus callosum (CC) are detected early in patients with probable MS. Methods: Brain MRI were performed in 28 subjects (20 females, 8 males) with a clinically isolated demyelinating event suggestive of MS (probable MS). 24 healthy subjects matched for age and gender served as controls. The mid-sagittal brain MR image was used for measurement of the CC (total area) and its sub-regions (anterior, middle, posterior). Results: The area of the CCn in patients with probable MS did not differ significantly from healthy subjects matched for age and sex. However, the total area of the CCn in the probable MS group significantly diminished from 539.6 ± 81.6 mm 2 at baseline, to 516.9 ± 57.3 mm 2 after one year (p < 0.05). The decrease was mainly attributed to the middle region of the CC that diminished from 227.0 ± 51.3 mm 2 at baseline to 209.9 ± 25.4 mm 2 after one year (p < 0.05). Our findings demonstrate that structural changes can be detected even in patients with probable MS, and thus support the role of early treatment even in patients with probable MS. PP14-01 DNA microarray analysis identifies CXCR3 and CCR2 ligand chemokines as early IFN-responsive genes in peripheral blood lymphocytes Jun-ichi Satoh a,b , Yusuke Nanri a , Wakiro Sato a , Takashi Yamamura a a Department of Immunology, National Institute of Neuroscience, NCNP; b Department of Bioinformatics, Meiji Pharmaceutical University, Tokyo, JapanObjective: To identify systematically interferon-beta (IFNB)-responsive genes in peripheral blood mononuclear cells (PBMC). Background: IFNB reduces the frequency of relapses and the number of new MRI lesions in RRMS. However, a proportion of the patients discontinue the treatment due to various adverse effects, most of which emerge at the early phase of the treatment. No biomarkers predicting detrimental responses of IFNB are available. Methods: Total RNA of PBMC incubated with 50 ng/ml rhIFNB in vitro was processed for cDNA microarray and real-time RT-PCR analysis. Results: Among total 1258 genes, IFNB elevated the expression of 107 and 87 but reduced 22 and 23 genes at 3 and 24 h. Upregulated genes were categorized into conventional IFN-response markers, components of IFNsignaling pathways, chemokines, cytokines, regulators of apoptosis and DNA damage, and heat shock proteins. IFNB markedly upregulated CXCR3 ligand chemokines (SCYB11, SCYB10 and SCYB9) active on effector Th1 cells, and CCR2 ligand chemokines (SCYA8 and SCYA2) effective on monocytes, whereas it downregulated CXCR2 ligand chemokines (SCYB2, SCYB1 and IL8) active on neutrophils. Eleven genes including ISG15, IFI6-16, IRF7, TAP1 and TNFAIP6 were elevated in 13 RRMS patients during IFNB treatment for 3 to 6 months. Conclusions: IFNB elevated immediately the expression of chemokines with relevance to early adverse effects in MS. PP14-02 Gene expression patterns identify short-term interferon-β-1a (Rebif) treatment effects in multiple sclerosis A. Achiron a,b , Y. Snir a , D. Magalashvili a , A. Feldman a , P. Sonis a and M. Gurevich a a Multiple Sclerosis Center, Sheba Medical Center, Tel-Hashomer, Ramat-Gan, Israel; b Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, IsraelIntroduction: Interferon-β-1a (Rebif) is one of the leading immunomodulatory drugs for the treatment of relapsing-remitting multiple sclerosis (RRMS). However, despite the well documented efficacy, it remains unclear what mechanisms are involved and how Rebif treatment alters the clinical course of the disease. Methods: For all experiments we used cDNA Affymetrix microarrays (U133A). We analyzed peripheral blood samples from 12 RRMS patients, before and 3 months after initiation of Rebif treatment. Data analysis was performed using t-test, TNoM (Threshold Number of Misclassification) and Info-test. Most informative genes (MIGs) were defined as those with pvalue <0.05 in all 3 statistical tests. Rebifinduced adhesion effects were characterized by down-regulation of the integrin genes like ITGA2B, ITGB3, ITGAE and ITGB5. Additionally, LIMS1, CD9, CD44 and SELP gene-transcripts, mediating cell adhesion, and TNFRSF4 that directly affects adhesion of activated T-cells to vascular endothelium, were suppressed. The anti-inflammatory effects of Rebif were mainly related to suppression of IL1 inflammatory-pathways and involved over-expression of IL1RN, and inhibition of CXCL5 and TOLLIP that are involved in IL1 activation. The anti-apoptotic genes like TGFB1, SNCA, GPX1 and IGF1R were down-regulated. Conclusions: Short-term Rebif treatment resulted in immunomodulatory effects related to anti-adhesion effects involving integrin family genes, suppression of IL1-related inflammation and stimulation of apoptosis, all important to control RRMS disease activity. PP14-03 Attaining RNA expression signatures in peripheral blood cells of interferon beta 1a (IM) treated multiple sclerosis (MS) patients over time This systematic time-course gene expression study focuses on the autoimmune disease MS and the effect of therapeutic intervention by Interferon-beta-1a (IFNβ1a). It is the aim to detect surrogate markers for drug-related molecular candidates in human PBMCs, to infer mechanisms of action affiliated to the effects/pathways of applied compound, and to discern RNA-signatures derived from (non-)responders to treatment.IFNβ therapy in patients with MS has proven efficacious, but appears suboptimal in terms of therapy responder rate. Large scale analysis of gene transcripts over time provide an unprecedented view of the complexity of studied disease and generates a huge data set that shall serve molecular group classifications.Employing the Affymetrix HGU133 set (33,000 genes) we perform an ongoing full genome study monitoring 20 patients receiving IFNβ1a (i.m., once weekly) over a period of 24 months. Samples were collected and directly processed before first treatment, after 1 week, 4 weeks, 1 year and will be drawn at corresponding dates. Systematic transcriptome analyses (supervised learning and literature-mining tools) are realized.Comparing time points, 115 genes showed increased or decreased expression levels in minimum 75% of the patients. 5 were IFN-modulated, 20 immune response-related, 5 identified in host defense and 3 in inflammatory responses.IFNβ1a may also alter the stability and coordination of cell components, as 30 genes were implicated in protein and lipid metabolism, cell adhesion and signaling, cytoskeletal (re-)arrangement and the ubiquitin pathway. The differential expression of the set of genes could be an important ancillary diagnostic tool. PP14-04 Longitudinal gene expression profiling in PBMCs of multiple sclerosis (MS) patients receiving Interferon-beta-1b therapy D. Koczan a , R. Goertsches a , S. Möller a , P. Serrano-Fernández a , HJ Thiesen a , UK. Zettl b a Institute of Immunology, University of Rostock, Rostock, Germany; b Clinic of Neurology, University of Rostock, Rostock, Germany A systematic time-dependent and comprehensive gene expression analysis from Interferon-beta-1b (IFNβ1b) treated MS patients will provide a rational basis for optimized or novel therapeutic strategies aimed at modulation of the disease course.Applying time-course microarray experiments to find new prognostic and diagnostic markers in peripheral blood cells after therapeutic intervention opens great perspectives regarding responsiveness or patient subclassification and has an ethical impact on the acceptance of costintensive treatment.Using the Affymetrix HGU133 set (approximately 33,000 genes) we performed a full genome study monitoring 18 patients over a period of 2 years. Samples were taken before first treatment and after 48 h, 4 weeks, 12 and 24 months. Only transcripts passing the detection threshold of the statistical algorithm MAS 5.0 were used for analysis. In order to distinguish molecular subgroups of treated patients based on generated RNA-profiles and clinical parameters, linear correlation models, supervised learning algorithms applying the bootstrap method and literature-mining tools were applied.Dynamic expression changes in individual RNA profiles could be ascertained; compared with pre-treatment expression level, 10 genes show differential expression at all time points. Introduction of profiling results into interaction exploring software generated valuable candidates (FCER1A; IFIT1, 3; HSXIAPAF1) for improved understanding of IFNb1b mechanism of action.The high proportion of IFN-regulated genes and the presence of MX1, a short-termed molecular indicator for IFNβ-activity, consistently induced in most of the patients support the validity of selected candidate genes. Being both immediately detectable and long lasting, the differential expression of these genes could be helpful for prospective purposes. PP14-05 Acute and steady state effects of interferon-beta in multiple sclerosis evaluated by gene expression profiling Treatment of multiple sclerosis (MS) with interferon (IFN)-beta modifies immune cell activation and cytokine secretion, adhesion molecules and chemokine receptor expression, and matrix metalloproteinases. We have recently found that the acute immunological effects of treatment with IFNbeta (i.e., within 24 h of drug injection) differ from the effects seen at steady state (i.e., more than 24 h after drug injection). We used the Affymetrix GeneChip Focus array with more than 8500 different genes displayed to study changes in gene expression in 10 patients treated with IFN-beta1a (30 μg intramuscularly once weekly). Blood samples for the study of mRNA expression in purified mononuclear cells (MNC) were drawn before the first injection of IFN-beta1a and 14-20 h after the first injection of IFN-beta1a (T1). After three months of treatment new blood samples were obtained before the injection of IFN-beta (T2) and after the injection of IFN-beta (T3). After the first injection of IFN-beta (T1) the expression of 283 genes differed significantly from baseline values (T0; p < 0.05 with Bonferroni correction). Among 135 up-regulated genes, 57 were induced at least two-fold. Among 148 down-regulated genes, 11 were at least 50% reduced. Gene expression at baseline and T2 did not differ significantly. After three months of treatment with IFN-beta, the injection of IFN-beta resulted in slightly less pronounced changes in gene expression, but there was no significant difference in the gene expression profile at T1 and T3. Treatment with IFNbeta results in acute changes but no major, persisting changes in blood MNC mRNA expression. PP14-06 In vitro inhibition of human CD4 + CD25 + regulatory T lymphocyte function by interferon-β through activation of autocrine/paracrine dopaminergic pathways Impairment of CD4 + CD25 + T lymphocyte (Treg) function might contribute to break down immune tolerance in patients with multiple sclerosis. We investigated in vitro the ability of Interferon β (IFN-β) to affect human Treg function as defined by production of IL-10 and TGF-β. Treg were isolated from healthy donors' blood by immunomagnetic sorting and cultured. IL-10 and TGF-β mRNAs and proteins were measured by RT-PCR and ELISA respectively. Incubation with IFN-β 1000 IU/ml significantly reduced IL-10 and TGF-β in Treg at both mRNA and protein levels (IL-10: mRNA = − 51.8 ± 12.0%, protein = − 35.3 ± 10.3%; TGF-β: mRNA = − 42.7 ± 16.7%, protein = − 46.9 ± 17.6%; P < 0.001 vs. control in all cases). As we recently reported that IFN-β induces the release of endogenous catecholamines from human lymphocytes, we first measured catecholamines in unstimulated Treg by HPLC-ED: we found elevated intracellular levels comparable to those in activated lymphocytes. We therefore tested whether the effects of IFN-β could be antagonized by selective antagonists for dopaminergic or adrenergic receptors: the D1-like receptor antagonist SCH23390 completely reversed the effects of IFN-β. The effect involves the activation of D1-like receptor pathways, likely through the autocrine/paracrine action of endogenous dopamine released from Treg by IFN-β itself. In view of the role or Treg in the regulation of the immune response in health and disease, further studies are urgently needed to assess the clinical significance of these findings. Introduction: Interferon β-1a (Rebif) is an effective treatment known to reduce disease activity and progression in patients with relapsing-remitting multiple sclerosis (RRMS). Using microarray gene-expression analysis we have recently identified 890 genes that characterize Rebif treatment effects in RRMS. Objective: Analysis of Rebif-induced regulatory pathways in order to further elucidate the hierarchy of biological functions associated with treatment effects in RRMS. Next, we identified common motifs of transcription factors in the promoter region of these regulating genes. Analysis of the motifs in the promoter region of the regulatory genes identified 48 transcription factors that added information for identification of interacting biological pathways induced by Rebif in RRMS. An interesting example for a previously unknown mechanism identified is the over-expression of zinc finger CCCH type domain containing 1 gene (ZC3HDC1). ZC3HDC1 was found directly regulate caspase recruitment domain family, member 4 gene (CARD4) that plays a significant role in apoptosis. PP14-08 Interferon-β1a-induced expression of brain-derived neurotrophic factor in human T lymphocytes Objective: To investigate the effects of interferon (IFN)-β1a (Rebif) on the expression of brain-derived neurotrophic factor (BDNF) and other neurotrophic factors in human T lymphocytes. Background: Neurodegeneration correlates with progression of disability in multiple sclerosis (MS). IFN-β1a reduces progression of sustained disability in MS, suggesting neuroprotective properties. BDNF and other neurotrophic factors capable of promoting neural cell survival may contribute to "neuroprotective immunity" in MS. Design/methods: For in vitro analyses, mitogen-activated peripheral blood lymphocytes (PBL) obtained from untreated MS patients or from healthy donors were treated with IFN-β1a. Their proliferative activity was compared to the secretion and transcription of BDNF as assessed by standard ELISA and by real-time PCR. Results: Proliferative activity of mitogen-activated PBL was inhibited by IFN-β1a in a dose-dependent manner. Highest BDNF levels were induced at IFN-β1a concentrations between 1000 and 5000U/ml, whereas higher or lower concentrations induced lower BDNF levels. BDNF mRNA levels, other neurotrophic factors and pro-and anti-inflammatory cytokines are currently being investigated. In addition, for ex vivo analyses, serum levels and mRNA levels of BDNF are being assessed in blood obtained from untreated or Rebif-treated MS patients. PP14-09 Modification of Interferon-β1b on CD4 + CD25 +high regulatory T cells and expression of FOXP3 mRNA in multiple sclerosis Shimizu, Y 1 , Ota, K 2 , Kawahata, K 3 , Ohara, K 1 , Ohashi, T 1 and Iwata, M 1 1 Department of Neurology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan; 2 Tokyo University of Science. Tokyo, Japan; 3 University of Tokyo Graduate School of Medicine, Tokyo, Japan Background: CD4 + CD25 + regulatory T cells have suppressive auto-reactive and potent regulatory properties. Especially, CD4 + CD25 +high regulatory T cells (Treg) are anergic to antigenic stimulation and actively downregulate activation. Recently, the transcription factor Foxp3 gene has been implicated as a key element in balancing immune responses and is expressed in CD4 + CD25 + Tr cells. The present study investigates whether Interferon (IFN)-β1b affects the inducing CD4 + CD25 high Treg cells and FOXP3mRNA expression. Method: We studied the frequency of CD4 + CD25 high Treg cells by flow cytometry analysis and the level of expression of FOXP3mRNA by quantitative real-time PCR in peripheral mononuclear cells (PBMC) from 18 healthy subjects and 18 multiple sclerosis (MS) patients before and after treating with IFN-β1b. Results: The frequency of CD4 + CD25 high Treg cells and the level of FOXP3 mRNA expression in PBMC showed no difference between healthy subjects and MS patients in the remission phase. The mean percentage of the frequencies of CD4 + CD25 high Treg cells in MS patients for 2 years after IFN-β1b treatment (2.7 ± 2.4%) were slightly higher than before treatment (2.0 ± 1.3%) (not statistically significant). Also an increase FOXP3mRNA expression was observed in MS patients at 3 months after IFN-β1b treatment (3.3 ± 3 .5 fold increases), it showed significantly different compared with before treatment (p < 0.05). Conclusion: Our results demonstrated that IFN-β1b might have played a role in controlling the auto-reactive T cells through an increased of Treg cells and also its relevance to the treatment of MS. PP14-10 The effects of interferon-β1b on expression of CXCR3 on circulating T cells in multiple sclerosis patients Background: Multiple sclerosis (MS) is an autoimmune disease in whose pathogenesis Th1 cells play an important role. It has recently become clear that interferon-β1b (IFN-β1b) treatment is effective in ameliorating relapsing-remitting multiple sclerosis (RRMS). The treatment efficacy of IFN-β1b for multiple sclerosis is potentially attributable to the immune regulatory properties of the drugs. Method: We compared the expression of Th1-related CXCR3 chemokine receptors and Th2-related CCR4 chemokine receptors on T cells derived from MS patients and those derived from healthy controls. Also, we have investigated the chemokine receptor expressions in MS patients undergoing IFN-β1b therapy. The expression of these Th1/Th2-related chemokine receptors were assessed at the baseline and longitudinally over a period of 24 months after the start of treatment in 10 RRMS patients grouped as 5 responders and 5 non-responders according to their clinical response to IFN-β1b therapy. Results: The percentage of CXCR3-expressing CD4 + T cells in patients with MS was significantly elevated compared with those of healthy controls. Treatment with IFN-β1b reduced the percentage of CXCR3-expressing CD4 + T cells in both responders and non-responders during the first 12 month. At 24th months after the treatment, the percentage of CXCR3expressing CD4 + T cells for responders was still reduced. However, that for non-responders returned to a level at baseline. Conclusion: the percentage of CXCR3-expressing CD4 + T cells would be a marker of immunological activity in MS patients, and IFN-β1b can correct the Th1/Th2 imbalance. PP14-11 Beneficial effect of interferon-β treatment in patients with multiple sclerosis is associated with transient increase in serum IL-6 level in response to interferon-β injection Objective: To elucidate whether untoward effects of IFN-β treatment such as headache and skin reaction assessed easily at bedside, and the background of each patient may have relationship with serum cytokine levels or not. In addition, we investigated if we could predict responder to treatment through these analyses. Methods: Twenty-five patients with multiple sclerosis (MS) before IFN-β 1b therapy were enrolled. Chronological blood sampling was performed 0, 10, and 24 h after injection of IFN-β. Body temperature, headache, arthralgia, and the size of skin reaction at the injected site were monitored for the first week. Serum levels of IFN-γ, TNF-α, IL-6, IL-10, and TGF-β were measured by enzyme-linked immunosorbent assay (ELISA), and the interactions between the change of cytokine levels and variables including headache, arthralgia, fever, size of skin reaction, age, EDSS scores at the initiation of the therapy and after one-year treatment, relapse rate before and after treatment, and type of MS were analyzed. Results: Transient increase in the serum IL-6 level in response to IFN-β administration correlates with headache, arthralgia, less relapse rate prior to the therapy, milder disability assessed by EDSS score at the initiation of the treatment, and less progression of disability during treatment. Conclusion: It may be helpful to assess serum IL-6 level at 2 points, before and 10 h after administration of IFN-β, for the prediction of the efficacy of IFN-β therapy. PP14-12 Analysis of neutralising antibodies in the Betaferon® in newly emerging multiple sclerosis for initial treatment (BENEFIT) study Neutralising antibodies (NAb) develop with all current immunomodulatory treatments for multiple sclerosis, but controversy exists about their clinical relevance. The BENEFIT study assessed the safety, tolerability and efficacy of interferon beta-1b (IFNB-1b; Betaferon®) 250 μg in patients with clinically isolated syndrome. Patients were randomised to IFNB-1b (n = 292) or placebo (n = 176) and treated for 24 months or until clinically definite MS (CDMS) was diagnosed. NAb for IFNB-1b were measured using the MxA assay on samples taken at baseline, every 6 months thereafter, and at end of study (EOS); NAb titres >=20 were considered positive. Additional analyses of the impact of NAb were performed focussing on patients with longer exposure to IFNB-1b, i.e. in patients with EOS after 180 days, 270 days and 360 days, respectively.The percentage of NAb-positive IFNB-1b-treated patients at each visit (month 6 to month 24) ranged from 16.5% to 26.1%. Reversion to NAbnegative status was observed in 22.7% of the NAb-positive patients. There was no significant effect of the presence of NAb on time to CDMS in any of the analyses performed: hazard ratios for NAb-positive versus NAb-negative patients were 0.63 (all IFNB-1b-treated patients), 1.01 (patients with EOS after 180 days), 1.14 (270 days) and 0.91 (360 days).In conclusion after 24 months on study, no effect of NAb on time to CDMS was detected. PP14-13 Qualitative differences of neutralizing antibodies against the different interferon beta preparations F. Deisenhammer, C. Gneiss, M. Reindl, T. Berger Department of Neurology, Innsbruck Medical University, Austria Interferon beta (IFNb) is a first line therapy for multiple sclerosis (MS). Three different types of IFNb are available, IFNb-1a for i.m. These three preparations have different potentials to induce neutralizing antibodies (NAB) which is not only related to different dosing and routes of application. Recognition of epitopes is one factor, where IFNβ-1a treated patients show significantly higher median binding titers against the N-terminal end of IFNβ than IFNb-1b treated patients (800 vs. 200) . Another factor is affinity, showing significantly higher affinity values for IFNβ-1a treated patients as compared to patients on IFNb-1b (296 +/− 50.7 vs. 265 +/− 31.5; values obtained by titration of antigen-antibody binding using increasing concentrations of sodium isothiocyanate). The potency of binding antibodies (BAB) to neutralize IFNb is also different among the different preparations. These biochemical characteristics contribute to the different abilities of the IFNb preparations to induce NAB and to their different dynamic of NAB development. Consequences for treatment decisions and therapy monitoring will be discussed as well as potential strategies to overcome NAB development or reversion to NAB negativity. In Multiple Sclerosis (MS), oligodendrocyte injury is believed to be caused by an aberrant immune response initiated by autoreactive T cells. Increasing evidence indicates that inflammatory responses in the central nervous system are not exclusively detrimental, but may also exert protective effects. Such protective effects are potentially mediated by the local secretion of neurotrophic factors by immune cells. We previously reported that T cells and monocytes produce leukemia inhibitory factor (LIF), a member of the neuropoietic family of neurotrophic factors, both in vitro and in inflammatory MS lesions. In the present study, we report a reduced LIF production by CD4 + T cells of relapsing remitting MS patient as compared to secondary progressive MS patients and healthy controls. Furthermore, immunomodulatory agents such as leptin, IFN-β and simvastatin were studied for their potential to alter secretion of LIF and other cytokines by T cells and monocytes. Low doses of simvastatin, but not IFN-β or leptin enhanced LIF secretion by CD4 + T cells of RR-MS patients, but not in healthy controls. We further demonstrated that LIF did not influence viability, proliferation and cytokine secretion of T cells. Together these data provide new information on the regulation of LIF secretion by immune cells. Further insights into the complex regulation of neurotrophic factors such as LIF may prove useful for treatment of MS. PP14-15 Identification and selection of disease related autoreactive T cells from early multiple sclerosis patients for specific T-cell vaccination CD4 + T cells sensitized against myelin epitopes are believed to play a role in the pathogenesis of MS. One experimental approach to treat MS, termed T cell vaccination (TCV), involves patient immunization with attenuated autoreactive T-cell lines responsive to myelin antigens.Distinguishing disease-related autoreactive lineages from normal T cell lineages reactive to myelin autoantigens in culture is essential for vaccine preparation. T-cell lines can also be generated from healthy subjects. Using microarray analyses we identified a unique autoimmune gene expression fingerprint of MOG-responsive T cell lines with over-expression of IGF-BP3, VEGF, BCL-2 and Lifeguard. This imprint is absent in myelinresponsive T-cell lines from healthy subjects. Over-expression of BCL2 suggested resistance of MS autoreactive lines to apoptosis. We thus protracted T cell exposure to antigen during line selection from the commonly used 16-18 days to ∼45 days, at which time normal human T cells undergo apoptosis. MS T cell lines generated were 93-99% CD4 + oligoclonal (4-9 TcR Vβ isoforms), IFNγ producing T cells bearing >90% CD45RO + determinants. We stimulated MOG responsive healthy subject T cell lines with the proinflammatory SDF1α, TNFα or IFNγ. Treated cells expressed higher levels of IGF-BP3, VEGF and BCL-2 genes mimicking the autoimmune fingerprint. Autoimmune fingerprint-bearing lines were insensitive to additional cytokine stimulation. While most P-MS patient lines express the autoimmune fingerprint and cannot be further stimulated, several MBP-reactive lines derived from P-MS bear healthy T cell features. This fingerprint enable distinguishing disease-related from healthy lineages within an MS or P-MS individual and enable better line selection for TCV. T cell lines reactive to synthetic myelin peptides were generated from peripheral blood of probable multiple sclerosis patients (P-MS), within three months of their first demyelinating attack. The attenuated autoreactive cell lines served as T cell vaccines in these patients. These cell lines were >98% CD4 + and >90% CD45RO + . Oligoclonal compositions of TcR Vβ family isoforms of 21 tested were identified. Vβ15 was present at a very high frequency in 9 of the 12 T-cell lines (75%) from 5 of the 6 patients (83.3%) which were examined. Vβ9 occurred in 8 of the 12 T cell lines (66.6%) from 5 of the 6 patients (83.3%) examined. Vβ7, Vβ5.1, Vβ6 and Vβ21 also occurred in higher frequencies of 50%, 42%, 42% and 30% respectively. It is noteworthy that the combination of Vβ9,15 was found in 5 of the 12 tested lines and the combination of Vβ 6, 7, 15, 21 in 5 other lines. Despite the apparent degenerate nature of the autoreactive TcR there appears to be common denominators in myelin antigens because stimulation of PBMC from these P-MS patients with a tetanus toxin epitope did not stimulate these autoreactive populations. Our findings indicate existence of Vβ clusters in P-MS patients which occur in repetitive combinations. These clusters are evident in T-cell lines generated in response to various myelin antigens within a given patient, irrespective of the nature of the specific antigen. PP14-17 T-cell vaccination in ms with CSF-derived activated CD4 + Tcells: Results of a placebo-controlled trial Activated anti-myelin T-cells accumulate in the cerebrospinal fluid (CSF) of MS patients, indicating that these T cells may represent a source of disease-related T cells. A previous pilot trial of T cell vaccination (TCV) with activated CD4 + T cells derived from CSF demonstrated safety, feasibility and immune effects in MS patients. A double-blind placebocontrolled trial was performed with early relapsing-remitting MS patients to study the effects of TCV on disease activity as measured by magnetic resonance imaging (MRI). Twenty-nine MS patients that demonstrated MRI activity in a pre-entry period of 6 months were randomized into active (TCV) (n = 20) and placebo (n = 9) groups. Three immunisations with irradiated CSF vaccines (5-10 million cells) or placebo were administered subcutaneously with an interval of 2 months. Patients were monitored for 12 months after the final immunisation for MRI activity (every 2 months), clinical scores and immune responses. The vaccinations were well tolerated and no toxicity or adverse effects were reported. Anti-myelin T cell responses were reduced after TCV in 16 out of 20 MS patients. The mean number of active MRI lesions and the volume of active MRI lesions and T2 lesions was reduced in the treated group, but not in the placebo group. These differences were however not statistically significant. The mean EDSS scores remained stable in both groups. Patients with a high immune response to the vaccine cells showed a trend for improved MRI and clinical responses to TCV. Despite the lack of statistically significant differences due to the low patient number, this study further demonstrates safety and feasibility of TCV in MS patients, and suggests that possible therapeutic effects of TCV may be more prominent in patients that show high immune responses to the vaccine. PP14-18 Immunoadsorption plasma pheresis therapy for the treatment of refractory attacks of multiple sclerosis T. Ohashi a , K. Ota b , Y. Shimizu a , K. Ohara a , C. Takeuchi a , M. Iwata a a Tokyo Women's Medical University, Tokyo, Japan; b Tokyo University of Science, Tokyo, JapanThe effectiveness of immunoadsorption plasma pheresis (IAPP) therapy for treating acute exacerbations of multiple sclerosis (MS) has not been well evaluated; thus, an accumulation of therapeutic experiences for the disease is important. In addition, IAPP has recently been reported to have a preventive efficacy, which added to its establishment as a preventive therapy. We report 4 MS patients with acute or subacute exacerbations. These patients have been unresponsive to intravenous methyl prednisolone (IVMP) therapy and intravenous immunoglobulin therapy but showed improvement after IAPP therapy. IAPP therapy was well tolerated and effective for a patient with acute exacerbation during pregnancy. Three MS patients who showed frequent exacerbations even after the introduction of interferon β-1b therapy had no relapses while undergoing periodical IAPP therapy that aimed to prevent relapses. Adverse events of IAPP therapy included deep venous thrombosis, catheter infection, and anaphylactic symptom. We concluded that IAPP therapy is a therapeutic option, especially for the patients with fulminant attack or with opticospinal form. It is relatively safe and applicable for patients with acute exacerbation during pregnancy. IAPP therapy could be evaluated further as an option for the prevention of relapsing-remitting MS in the near future. PP14-19 IVIg decreases matrix metalloproteinase-9 production, not tissue inhibitor of metalloproteinase-1, by PBMC of multiple sclerosis patients K. Okada a , S. Tsuji a a Department of Neurology, University of Occupational and Environmental Health., Kitakyushu, JapanObjective: To determine whether IVIg decreases matrix metalloproteinase (MMP)-9 production by peripheral blood mononuclear cells (PBMC) of multiple sclerosis (MS) patients. Methods: PBMC were prepared from relapsing and remitting MS patients (n = 10), who were not administered corticosteroid within 90 days or have never been introduced with interferon-β, and the healthy control (n = 10). PBMC were incubated with or without lipopolysaccharide (LPS, 1 μg/ml) for 1 h, and then IVIg (0.1-10 mg/ml) was added for 24 h. Moreover, to study the neutralizing effect of IVIg on MMP-9, PBMC were incubated with LPS (1 μg/ml) for 24 h, and then IVIg (0.1-10 mg/ml) was added for 1 h. MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 in the supernatant were measured using ELISA. Statistical analysis was performed using Mann-Whitney U test. Result: MMP-9 was significantly higher in the MS group than the control group both in the condition with and without LPS. Although TIMP-1 was up-regulated with LPS in both of the two groups, there was no significant difference between the two groups. The MMP-9/TIMP-1 ratio was significantly higher in MS group than the control group. Suppressive effects of IVIg on MMP-9 production were not by neutralizing effects. PP14-20 The changes of serum cytokines in the treatment of IAPP for Multiple Sclerosis Ohji S, Tomioka R, Mitsui T, Yoshida N, Iguchi T, Takahama M, Ohnuki M, Nomura K Saitama Medical Center, Department of Neurology, Saitama Medical University, Saitama, Japan[Purpose] The immuno adsorption plasma pheresis (IAPP) has recently become available for the treatment of MS. However, the mechanism of IAPP is still unclear. In this study, we measured serum cytokines to investigate the mechanism of immuno-modulation effects of IAPP. [Materials and methods] IAPP was carried out in 4 patients with MS in the active stage, that could not have enough clinical improvement with steroid therapy. Serum cytokines 2, 4, 5, 6, 8, 10, 12, IFNgamm, were determined by using Beads Array Method (BD Bioscience Co.), in both outflow and inflow of IAPP circulation, and in the outflow of circulation just before and after the treatment. [Results] In the study of the changes of cytokines in both outflow and inflow while a treatment, IL-6, 8, 10 decreased in inflow, comparing to outflow. Also, in the outflow of circulation just before and after an IAPP, IL-6, 8, 10 increased just after IAPP in all 4 cases. [Conclusion] The adsorption column removed various cytokines nonspecifically. IAPP for MS stimulated the production of mainly Th2 cytokine IL-10, and made immuno-state shift to Th2 balance. PP14-21 Natalizumab effects on immune cell responses in multiple sclerosis Objective: To study in vivo biological effects of natalizumab on immune cell phenotype and function in MS patients. Background: While natalizumab holds promise as an effective therapy for MS, the emergence of cases of PML creates the imperative to better define mechanisms underlying both beneficial as well as adverse drug effects. Development of simple assays that measure these effects could prove useful in immune monitoring of patients on this emerging therapy.Methods: Blood was obtained before and after serial monthly natalizumab infusions to track functional expression of VLA-4 and migratory capacity of immune cells. The impact of infusion on activation thresholds of immune cells was evaluated. Results: Pre-infusion VLA-4 expression differed across immune cell subsets. Natalizumab significantly, albeit partially, diminished VLA-4 expression on circulating immune cells. Treatment significantly decreased migratory capacity of immune cells, correlating well with changes in VLA-4 expression. Effects of a single dose were not saturating and did not persist through the monthly dose-interval. Infusion effects varied across patients but were remarkably stable within individual patients, over multiple infusions. Treatment significantly modulated proliferative responses of immune cells. Interpretation: We provide first proof-of-concept that in vivo natalizumab diminishes migratory capacity of human immune cells. Our prospective study shows that effects of therapy likely: (i) differ for distinct immune cell subsets, (ii) are not sustained over current dose interval, (iii) have unique profiles in individual patients, and (iv) include modulation of activation thresholds of immune cells. Monitoring these parameters could be relevant to ongoing safety and efficacy considerations. Objective: To assess safety and immune modulation by BHT-3009 (a DNA plasmid that expresses full-length human MBP) in MS patients. Within this trial we are measuring several immune parameters including CFSE based antigenspecific T cell proliferation and intracellular cytokine production by PBMCs. There were 22 treatment-related adverse events (AEs), all of which were mild/moderate: 12 on placebo and 10 on BHT-3009 arms.Data from the CFSE assay on four patients from two centers who ran this assay in the first two cohorts demonstrate antigen-specific reduction in MBP reactive T cells by week 9 of treatment in three of them. In the first patient, the proliferation of MBP83-99 reactive IFN-γ positive cells decreased from 25.9% to 1.2%. In a second patient, the proliferation of MBP83-99 reactive IFN-γ positive cells decreased from 13.3% to 5.4%. In the third patient, the proliferation of whole MBP reactive IFN-γ positive cells decreased from 2.27% to 0.79%. In all three of these patients the proliferation to tetanus toxoid did not decrease, pointing to antigen specificity following BHT-3009 treatment. In a fourth patient, there was no change in either the MBP83-99, whole MBP or tetanus proliferative responses. Conclusion: The data indicate that BHT-3009 is safe and may suppress immune responses in an antigen-specific manner. Background: Anti-tumor necrosis factor α (anti-TNFα) therapy, including infliximab treatment, for rheumatic diseases has been associated with rare cases of new or exacerbated neuroinflammatory disorders such as multiple sclerosis and optic neuritis. In multiple sclerosis, anti-TNFα therapy may increase disease activity. There is evidence for an increased peripheral T cell reactivity, measured as enhanced IFNγ production, during anti-TNFα therapy of rheumatoid arthritis. This may potentially provoke neuroinflammation, since systemic IFNγ administration has been demonstrated to exacerbate multiple sclerosis. We therefore hypothesize that the few cases of clinical neuroinflammatory disorders observed after anti-TNFα therapy of rheumatic diseases represents the extreme end of a commonly occurring minor intrathecal immune activation, which in most cases does not give any overt neurological dysfunction. Objective and methods: To test this hypothesis, we determined the expression of IFNγ, TNFα and IL-10 mRNA in cerebrospinal fluid cells and peripheral blood mononuclear cells, the levels of nitric oxide oxidation products in cerebrospinal fluid and serum, the cerebrospinal fluid cell counts and IgG indices, in ten patients with polyarthritis before and during infliximab treatment. Results: No significant signs of intrathecal immune activation were recorded. In the systemic compartment, induction of IFNγ expression during infliximab treatment was demonstrated. Conclusions: Intrathecal immune activation, as measured by the employed assays, during infliximab therapy is not a common phenomenon and we thereby refute our initial hypothesis. Our finding of increased systemic IFNγ expression demonstrates that a systemic pro-inflammatory component is part of the diverse effects of anti-TNFα therapy. PP14-24 Statins inhibit neuronal cell injury promoted by activated T lymphocytes Background: Cell-cell contact interactions play a major role in promoting neuronal cell injury in neuroinflammatory diseases. Activated T lymphocytes cause injury in a cell contact-dependent non antigen-restricted manner. We hypothesize that statins could offer neuroprotection by down regulating T-cell activation, cell-cell contact-dependent interactions and matrix metalloproteinase (MMP) expression. Methods: Statin-and control-treated peripheral blood lymphocytes were co-cultured with two neuronal (SHSY-5Y and NB-1) and one astrocytic cell line (LN18). Functional antibodies directed against CD147, CD98, HLA-DR and HLA-ABC attenuating T-cell activation were pulsed on T cells and then presented to target cells. Neurons were also incubated with different proinflammatory cytokines and chemokines. Cell functions were assessed by MTT assay, microscopy, ELISA, FACS and zymography. Results: Neurons were highly susceptible to cytotoxicity caused by activated T cells. Neuronal cell viability was restored when T cells were pretreated with statins (which again was reversed by mevalonate) and also when prepulsed with T-cell activation antibodies (CD147 and CD98 but not HLA-DR and HLA-ABC). Proinflammatory cytokines and chemokines did not promote neuronal apoptosis. Further we observed a decrease in MMP-9 in statin-treated cultures. Cell apoptosis was specific to neuronal cell lines as the astrocytic cell line did not show a similar behaviour. Conclusion: Treatment with statins down regulates cell activation markers which mediate the neuronal insult. Cell death on neurons is to be further reconfirmed by MAP-2 immunoreactivity on neurons and detection of nucleosomes in culture medium. Thus, statins may promote neuroprotective properties in neuroinflammatory disorders such as multiple sclerosis. PP14-25 Lymphocyte homeostasis and regulatory cells in multiple sclerosis The hallmark of autoimmune diseases such as multiple sclerosis (MS) is the breakdown of self-tolerance. Naturally occurring regulatory T cells (Tregs), which preferentially express high levels of CD25 and the transcription factor forkhead box P3 (FoxP3), are critical to the maintenance of peripheral tolerance and prevention of autoimmune disease. In MS patients Treg numbers are normal but they are functionally impaired compared to healthy controls.We have treated a cohort of patients with MS using Campath-1H, a monoclonal antibody that induces a profound and prolonged T cell lymphopaenia. In the first few months after treatment, there is a five-fold increase in serum interleukin-7 (IL-7), a cytokine that promotes homeostatic proliferation, and an over-representation of cells expressing high levels of CD25 in the depleted T cell pool. We sought to determine whether these were truly Tregs using a functional assay involving the fluorescent dye carboxy fluoroscein diacetate succinimidyl ester (CFSE). IL-7 is reported to induce the transient expression of high levels of CD25 on CD4 + cells; however, these cells do not have suppressor cell function. We investigated the role of IL-7 in the genesis of these CD25 hi cells; spiking healthy control human peripheral blood mononuclear cells (PBMCs) with increasing concentrations of recombinant human IL-7 resulted in an increase in the percentage of CD4 + cells that express high levels of CD25, as determined by flow cytometry.In the post-Campath-1H lymphopaenic patient, IL-7 appears to link homeostatic mechanisms driving lymphocyte reconstitution with the enrichment of putative regulatory T-cells. PP14-26 Neurotrophin secretion after lymphodepletion with Campath-1H in multiple sclerosis Multiple sclerosis (MS) is a chronic neurological disorder thought to be driven by auto-reactive T lymphocytes, resulting in acute inflammatory axonal transaction, focal demyelination and oligodendrocyte destruction, with subsequent chronic axonal loss.The therapeutic monoclonal anti-CD52 antibody, Campath-1H, induces a prolonged T lymphopaenia. Treatment of patients with early, aggressive, relapsing-remitting MS results in a 94% reduction in relapse rate and a sustained improvement in fixed disability (as assessed by the Expanded Disability Severity Score). Such reported efficacy is unparalleled in trials of other MS therapies. Early improvement may represent abrogation of inflammation-associated conduction block, but this fails to explain sustained improvement seen at 12 to 24months.We hypothesise that growth factors are secreted in the context of immune reconstitution after Campath-1H treatment, and that these may promote neuronal survival and repair.ELISA (Enzyme-Linked Immunosorbant Assay) was used to determine concentrations of neurotrophins in peripheral blood mononuclear cell (PBMC) culture supernatants pre-and post-Campath treatment. Our results show that patients' PBMCs secrete neurotrophic factors after Campath-1H treatment: we found an increase in Ciliary Neurotrophic Factor (CNTF), a substance recently shown to enhance myelin formation; an even greater elevation in Brain Derived Neurotrophic Factor (BDNF); but a dramatic reduction in insulin-like growth factor-1 (IGF-1) secretion.We explore the functional significance of these observations using a well established in vitro model, in which neurons derived from rat embryonic cortices are cultured and exposed to PBMC conditioned media. Untreated MS patients had a greater proportion of mature naïve (CD27 − 23 + ) and fewer memory (CD27 + ) B cells compared with healthy controls. One month after Campath-1H, there was a profound lymphopenia. By three months, the total number of B cells had recovered, while T cell numbers remained depleted even at twelve months post treatment. Within the B cell population, mature naïve cells remained the dominant cell type, but memory cells were further decreased, in contrast to the relative dominance of memory cells in the depleted T cell pool. One month after treatment the proportion of transitional type 1 (CD27 − / CD23 − ) B cells, the recent bone marrow emigrants, increased dramatically, suggesting they are principally responsible for the reconstitution. Also at one month, serum B cell activating factor (BAFF) increased significantly; but by month three levels had dropped and we observed a switch from T1 to mature naïve B cell phenotype. This is in agreement with published work showing the importance of BAFF in naïve B cell maturation and survival. However, ex vivo neutralisation of BAFF did not alter B cell proliferation in our patients. PP14-28 Differential expression of FOXP3, CTLA-4 and GITR gene in CD25 + CD4 + T regulatory cells before and after corticosteroid treatment during relapses in interferon treated multiple sclerosis patients CD25 + CD4 + T regulatory (TR) cells seem to play a crucial role in the maintenance of immunological self-tolerance in autoimmune diseases such as multiple sclerosis (MS). Recent experimental data have shown that Forkhead box P3 (FOXP3) is highly expressed in TR cells and is responsible for their function. Cytotoxic T lymphocyte antigen 4 (CTLA-4) is constitutively expressed in TR cells; it is responsible for the costimulation of TR cells and the direct suppression of effector T cells. Glucocorticoid induced TNFRSF 18 (GITR) is upregulated in TR cells and its triggering leads either to apoptosis or proliferation of TR cells.In this study we measured the mRNA and protein levels of FOXP3, CTLA-4 and GITR in TR cells in interferon treated relapsing-remitting multiple sclerosis patients before and after corticosteroid treatment. All patients were examined according to expanded disability status scores (EDSS) to be in relapse. CD25 + CD4 + T cells were isolated from PBMCs and total RNA was extracted. The quantification of FOXP3, CTLA-4 and GITR mRNA level was performed by Real-time PCR. The quantification of protein level was performed by Western blot followed by densitometry. All patients have been tested for antibody mediated neutralizing activity against interferon-beta molecule. Our results indicate a clear trend in which corticosteroid treatment results in upregulation of GITR molecule. PP14-29 Immunomodulatory therapies and low dose Azathioprine reverse partially and significantly visual and neurological deficits in progressive multiple sclerosis Prince of Wales Hospital, Sydney, Australia Objective: To determine the effect of the addition of low dose Azathioprine to immunomodulatory therapies in progressive demyelinating disease. Methods: Azathioprine 50 mg daily was added to an immunomodulatory therapy in patients with progressive multiple sclerosis and progressive demyelinating optic neuropathy. Patients were observed for twenty four to forty eight months. Results: Partial and significant reversals of deficits in cognition, visual acuity, colour vision, balance, strength, sensation, and micturition followed the addition of low dose Azathioprine to immunomodulatory therapies in all patients. Reversals in visual acuity were significant (p = 0.016) as were reversals in colour vision (p = 0.009).Vision in the patient with progressive demyelinating optic neuropathy of five years improved after the commencement of "combination therapy", deteriorated when either the immunomodulator or Azathioprine was withdrawn and improved when the "combination" was reinstituted. The effect of "combination therapy" upon visual acuity was significant (p < 0.001) as was the effect on colour vision (p < 0.005) and has been maintained for two and a half years. Conclusion: The addition of low dose Azathioprine to immunomodulatory therapies may ameliorate visual and neurological deficits in progressive multiple sclerosis. Therapy instituted in the early phase may diminish progression and lessen disability. Visual acuity and Ishihara scores may be robust in vivo "therapeutic markers". The long term adverse consequences of therapy remain to be determined. PP14-30 The double edged sword of treating autoimmunity: Insights from therapeutic lymphocyte depletion in the treatment of multiple sclerosis Two consequences of the treatment of multiple sclerosis with the lymphocyte depleting monoclonal antibody Campath-1H have been observed; prolonged T cell lymphopaenia and the emergence of novel autoimmune diseases. It has been shown that this coincides with a period of lymphopaenia induced proliferation (LIP). Animal models of autoimmune disease employ LIP to induce susceptibility to disease, suggesting that homeostatic proliferation inherently results in the loss of self tolerance. The treatment of multiple sclerosis offers a unique opportunity to study this phenomenon in humans. We have conducted a survey of all autoimmune diseases arising during the treatment of multiple sclerosis with Campath-1H at our institution. The autoreactivity of lymphocytes prior to and following this treatment was measured by challenging carboxy fluoroscein diacetate succinimidyl ester labelled PBMCs with the putative autoantigens myelin basic protein and thyroid stimulating hormone receptor extracellular domain. We report an incidence of autoimmunity after treatment with Campath-1H of 39%. The incidence was greatest in those patients receiving a single pulse of Campath-1H, a group who were also older and had more advanced multiple sclerosis. A variety of autoimmune conditions have been identified, including organ specific, haematological and systemic autoimmunity; the most prevalent was thyroid autoimmune disease. An eightfold increase in the autoreactivity of lymphocytes emerging during LIP, which resides within the CD4 + T cell compartment, is demonstrated. We hypothesise that this may, at least in part, be responsible for the emergence of autoimmunity in patients undergoing lymphocyte depletion for the treatment of multiple sclerosis. PP14-31 Simvastatin affects microglia cell motility Statin treatment is proposed to be a new potential therapy for multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system. Although their anti-inflammatory properties have been studied extensively, the effects of statins on brain cells, however, are until now poorly investigated. We therefore evaluated the effects of simvastatin treatment on the migratory capacity of brain microglial cells, key elements in the pathogenesis of MS.Using FACS analysis, we show that exposure of human and murine primary microglial cells to simvastatin reduces cell surface expression of the chemokine receptors CCR5 and CXCR3. In addition, simvastatin treatment specifically abolishes chemokine-induced microglial cell motility, as determined by chemotaxis assays.Furthermore, using cytoskeleton staining and structural electron microscopy we show that simvastatin treatment of microglia alters actin cytoskeleton distribution and leads to changes in intracellular vesicles. These data clearly show that simvastatin inhibits several immunologically relevant features of microglia, which may provide a rationale for statin treatment in MS.Supported by the Dutch MS Research Foundation (grant 00-407 MS).Background and objective: Few patients with multiple sclerosis (MS) were more than 65 years old. According to McDonald's diagnostic criteria for MS, the criteria would best apply to patients between 10 and 59 years of age and special care must be taken in making a diagnosis of MS in those who were younger or older. Although some cases of late-onset MS were reported, clinical characters of late-onset MS were still unknown. Therefore we evaluated characteristics of late-onset MS in our hospital. Late onset is defined as onset after 65 years old and we found out three patients (68-77 years old, one woman and two men). Clinical course and MRI findings of these patients were evaluated. Results: All three patients with late-onset MS underwent relapse and remission. The sites of the lesion were optic nerve and spinal cord in one patient, cerebrum and spinal cord in two patients. All their symptoms were temporarily improved after steroid therapy. Two patients with late-onset MS had poor prognosis, but in one patient, subcutaneous administration of Interferon β1b was effective for preventing relapse for 2.5 years. Conclusion: Characters of late-onset MS were similar to Japanese ordinary type of opticospinal form. Interferon β1b therapy may provide good prognosis in late-onset MS. PP15-03 Questionnaire on the expediency of DNA microarray analysis for differential diagnosis of multiple sclerosis and prediction of therapeutic response to interferon-beta Yusuke Nanri, Jun-ichi Satoh, Wakiro Sato, Takashi Yamamura Department of Immunology, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, JapanObjective and methods: The accurate diagnosis of multiple sclerosis (MS) is often hampered by a difficulty in differential diagnosis of neurological diseases mimicking MS, such as Sjögren syndrome (SjS) and neuro-Behçet's disease. By gene expression profiling of peripheral blood T cells on a DNA microarray, we recently found that MS is classified into four distinct subgroups with different therapeutic responses to interferon-beta (IFNB), separated from healthy control subjects (Satoh et al. To determine the expediency of DNA microarray analysis for a support in differential diagnosis of MS and prediction of IFNB response in clinical practice, an 18-item questionnaire was faxed to certified neurologists of 1709 hospitals in Japan.Results: The answer rate was 19% (319/1709). Great numbers (54% and 56%) of neurologists experienced difficulties in discriminating MS from tumors and cerebrovascular accidents, where 70 and 4 cases required pathological diagnosis, respectively. Nine neurologists suggested difficulties in differential diagnosis of MS and SjS. Approximately a half (46%) of IFNB-treated MS patients showed satisfaction with the treatment, while 25% of the patients were grouped into poor responders, composed of dropouts due to the induction of severe relapses or adverse effects. The great majority (86%) of neurologists showed a favorable answer to the expediency of DNA microarray analysis for prediction of IFNB response. Conclusions: Even by certified neurologists, the accurate diagnosis of MS is often difficult. The potential power of gene expression profiling of peripheral blood T cells makes most neurologists to have the expediency of DNA microarray technology for differential diagnosis of MS and prediction of therapeutic response to IFNB. PP15-04 Coincidence of multiple sclerosis and autoimmune diseases Background: Multiple sclerosis (MS) could be associated with other autoimmune diseases (ADs). The aim of our study was to quantify the coincidence MS with an ADs and to characterize the MS with other AD. Patients and methods: Data of 194 patients with definite diagnosis of MS (134 women and 60 men, aged 36.5 ± 9.1 years, range 18-62 years, disease duration 8.6 ± 6.4 years), defined by the Poser criteria, were retrospective analysed. The two groups -MS with and without ADs were compared. 14 (7.2%) of the patients with MS had other AD. The most frequent ADs were: autoimmune thyroiditis 7, uveitis 2, rheumatoid arthritis 1, ulcerative colitis 1, coeliac disease 1, asthma bronchiale 1, psoriasis vulgaris 1, spondyloarthritis 1 (one patient had autoimmune thyroiditis and rheumatoid arthritis). These ADs preceded MS diagnosis and interferon beta therapy in all cases. There were found no significant differences in the age and EDSS between MS group with and without ADs. Conclusion: Our results showed similar findings as other reports. Neurologic disability is not significantly influenced by coincidence MS and other AD. Autoimmune thyroiditis showed the strongest coexistence with MS, but this may reflect the high prevalence in common population. PP15-05 Two cases of biopsy-proven tumefactive multiple sclerosis Yoshii S, Iizuka T, Masuda R, Ujiie S, Hamada J, Sakai F Department of Neurology, School of Medicine, Kitasato University, Sagamihara, JapanObjectives: to report two cases of steroid-resistant tumefactive multiple sclerosis (MS), which improved on combination therapy including repeated steroid pulse, intravenous immunoglobulin (IVIG) or plasma exchanges (PE). Methods: Case reports.Results: the first case is a 21-year-old woman who presented with convulsive seizure following the onset of headache and fever. She underwent brain biopsy, which showed abundant accumulation of lipid-laden macrophages with perivascular lymphocytes compatible with acute MS. She did not improve following steroid pulse therapy, but clinical symptoms and brain MRI findings gradually became stabilized after repeated combination therapy consisting of steroid pulse and IVIG, followed by interferon beta. He was admitted to another hospital where under a diagnosis of brain tumor he underwent open brain biopsy, which showed no malignancy. After given a diagnosis of MS, he received steroid pulse therapy without beneficial effect. He also received steroid pulse therapy followed by IVIG at the second hospital, but he progressed relentlessly and became bed-ridden. He was transferred to this hospital. He initially received IVIG with steroid pulse therapy because he was in a septic state. He underwent PE followed by interferon beta. His neurological deficits remained unchanged but the activity of MS on MRI markedly decreased. Repeated combination therapy may be effective if began during earlystage of MS. PP15-06 A case report of the patient with tumefactive demyelinating lesion masqueraded brain tumor M. Apiwattanakul Prasat Neurological Institute, Bangkok, ThailandObjective: Tumefactive demyelinating lesion is quite rare in clinical practice. Surgical biopsy is usually performed to differentiate between two conditions. Method: A case report of Thai young man who presented with progressive left hemiparesis and right side dullness headache in the past two months.Magnetic resonance imaging and surgical biopsy were done to demonstrate his pathology. Result: MRI revealed hyposignal T1 weighted/hypersignal T2 weighted and Fluid-Attenuated Inversion Recovery (FLAIR) at right frontoparietal white matter area with gadolinium enhancement in the lesion which some of these were not completed rings. There is also another lesion at left parietooccipital area but lesser in size and enhancement. Magnetic resonance spectroscopy (MRS) shown dropping of N-acetyl aspartate (NAA) peak with modest increased in choline (Cho)/phosphocreatine (Cr) ratio which was compatible with demyelinating disease. Histopathology demonstrated foamy macrophage, perivascular lymphocytic infiltration, prominent reactive astrocyte and special stains revealed markedly loss of myelin with myelin debris. The patient was diagnosed with tumefactive demyelinating lesion and his clinical status was improved after high dose steroid administration. Conclusion: In patient who presents with progressive neurological deficit and mass liked lesion in neuroimaging, in addition to tumor such as glioma, a variant form of demyelinating disease such as tumefactive demyelinating lesion should be in differential diagnosis. But biopsy should be always done to discriminate between two conditions because of difference in treatment and prognosis. PP15-07 Three cases of relapsing multiple sclerosis presenting with only nausea and vomiting H. Fukaura a , S. Takahashi a and Y. Terayama a a Neurology, Iwate Medical University, Morioka, Japan Two male and one female patients with multiple sclerosis (MS) were admitted to our hospital because of nausea and vomiting. Upper gastroenterological examination showed no abnormalities. After the treatment of intravenous methylprednisolone sodium, nausea and vomiting improved.Lesions involving the dorsal motor nucleus of the vagus, the nucleus ambiguous or the autonomic nuclei in the medullary reticular formation are usually associated with vomiting and upper gut motility disturbances. We believe our cases represent an unusual manifestation of MS. PP15-08 Depression score in multiple sclerosis patients Ohara, K 1 , Ota, K 2 , Shimizu, Y 1 , Ohashi, T 1 and Iwata, M 1 1 Department of Neurology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan; 2 Tokyo University of Science, Tokyo, JapanBackground: Some patients of multiple sclerosis (MS) have psychic symptoms. Depression is observed as one of the frequent psychic symptoms of MS. However, many papers, researched psychological interventions for MS, were published, but they did not form any definite conclusions. To investigate MS depression, 27 MS patients (7 males, 22 females; 24 relapse and remitting type, 2 secondary progressive type, 3 with clinical isolated syndrome; average age: 43) were compared and categorized or examined with respect to age, depressive score, EDSS, morbidity period, those receiving interferon beta 1b therapy and those who did not, total frequency of relapse, and frequency of relapse the last year. Results: There were no significant differences. Between depressive score and EDSS, numbers of cerebral lesions, total frequency of relapse, frequency of relapse in the last year, among patients with high EDSS. PP15-09 Characterization of the T cell receptor repertoire in neuromyelitis optica: T cell activity is up-regulated compared to multiple sclerosis Y. Warabi a,b , K. Yagi a , H. Hayashi a and Y. Matsumoto b a Tokyo Metropolitan Neurological Hospital, Tokyo, Japan; b Tokyo Metropolitan Institute for Neuroscience, Tokyo, JapanTo characterize T cell immunity in neuromyelitis optica (NMO), we examined the T cell receptor (TCR) repertoire in NMO patients with complementarity-determining region 3 (CDR3) spectratyping and compared the results with those from multiple sclerosis (MS) patients and healthy subjects. From 11 patients with NMO, 21 MS patients and 25 healthy subjects, peripheral blood lymphocytes (PBL) were isolated.Written consent was obtained from all the subjects and the study was approved by the Institute Review Board. RNA was extracted from PBL and cDNA was synthesized, then, amplified using primer pairs for TCR to determine CDR3 spectratypes. Both NMO and MS patients had a larger number of clonally expanded Vβ genes than healthy subjects. Moreover, NMO patients had a significantly larger number of expanded Vβs than MS patients. The detailed analysis revealed that Vβ1 and Vβ13 were significantly activated in NMO. These results reflected unique pathophysiology of NMO, which is distinguishable from that of MS. Furthermore, longitudinal examinations of the TCR repertoire demonstrated that the number of clonally expanded Vβs in NMO correlates with the Kurtzke Expanded disability status scale (EDSS). Although the activation pattern of the TCR repertoire in relapsing-remitting MS (RRMS) was similar to that in NMO, secondary progressive MS (SPMS) patients with longer disease durations and higher EDSS scores consistently had a smaller number of clonally expanded Vβs than RRMS patients. Detailed TCR investigations will provide useful information to evaluate the clinical and immunological status of NMO and MS and to develop effective immunotherapies. PP15-10 Brain MRI findings in Japanese MS patients with NMO-IgG I. Nakashima, K. Fujihara, I. Miyazawa, T. Misu, and Y. Itoyama PP15-11 Large "cystic" cerebral lesions in multiple sclerosis patients after discontinuation of IFNβ-1b: A report of two cases T. Takahashi, I. Nakashima, T. Misu, Y. Shiga, K. Fujihara, Y. Itoyama Department of Neurology, Tohoku University School of Medicine, Sendai, Japan Interferon (IFN) β is an established disease-modifying therapy for multiple sclerosis (MS). However, the following course after discontinuation of IFN-β is not fully known. Here we report two patients who developed large "cystic" cerebral lesions after IFNβ-1b was discontinued. Although pathogenetic mechanism of developing cystic cerebral lesions remains unknown, our cases suggest that discontinuation of IFNβ might be linked to the development of the cystic lesions. In order not to overslip such lesions after discontinuation of IFNβ-1b, we should check brain MRI regularly or when any cerebral symptoms are noticed regardless of severity. PP15-13 Analysis of the opticospinal, conventional forms of multiple sclerosis, and neuromyelitis optica in Taiwan Jen Jen, Su; Chih Chao, Yang Department of Neurology, National Taiwan University Hospital, Taiwan Multiple sclerosis (MS) is an inflammatory demyelinating disease of unknown etiology, and increasing evidence suggests that it is heterogeneous. On the other hand, concerning the sites of involvement, two further MS subtypes in Japan were separated into opticospinal MS (OS-MS), in which the clinically estimated main lesions are confined to the optic nerves and spinal cord, the conventional MS (C-MS), which shows disseminated lesions in the central nervous system (CNS), including the cerebrum, cerebellum or brainstem.Neuromyelitis optica (NMO) is an inflammatory demyelinating disease that selectively affects optic nerves and spinal cord. A specific IgG for NMO was described in NMO and Asian optic-spinal form of MS, but not detected at classical (western) form of MS. But whether OS-MS is NMO? There were still many debates and discussions.In Taiwan there were increasing numbers of multiple sclerosis and there were many patients presenting optic nerve and spinal cord involvement but less brain involvements. We try to evaluate the patients of multiple sclerosis admitted at one primary medical center in Taiwan, to classify them into OS-MS, conventional MS (C-MS) and NMO then to analyze whether there were differences between their clinical course, prognosis and neuroimage studies and tried to correlate the relationship of OS-MS and NMO in Taiwan. Neuromyelitis optica (NMO), or Devic's disease is a disorder of the demyelinating disease spectrum which involves mainly the optic nerves and the spinal cord within the central nervous system (CNS). Recently, abnormalities of the humoral immune system have been shown in cases with NMO, with an antibody against aquaporin-4. Here we aimed to look for NMO IgG in Turkish cases with Devic's disease in comparison to cases with classical multiple sclerosis (MS) and healthy controls (HC). Serum samples from 14 patients with Devic's disease, 14 cases with MS and 15 HC were assessed using an indirect immunofluorescence kit containing monkey cerebrum, cerebellum, intestinum and HEP2 cells. The sections were assessed blinded to the clinical diagnosis. 8 patients with Devic's disease showed staining around the vessels and pial surfaces in cerebrum and cerebellum sections. On the other hand none of the MS cases or HC showed such a staining. 5 out of 8 positive Devic's showed strong binding, and among these 4 had severe clinical involvement. On the other hand, among the cases with Devic's disease that were negative, 2 had severe disease while 4 had milder forms of the disease. However this difference did not reach statistical significance. This relatively small cohort of patients suggest that the presence of NMO IgG might have an association with the disease course and severity. Such a relationship should be further assessed in larger series. PP15-15 Acute disseminated encephalomyelitis in Japan: A population-based study of pediatric patients General Hospital, Omuta, Japan; e University of Occupational and Environmental Health, Kitakyushu, Japan; f Kurume University, Kurume, Japan; g Kitakyushu City Yahata Hospital, Kitakyushu, Japan; h Aso Iizuka Hospital, Iizuka, Japan; Nippon Steel Yawata Memorial Hospital, Kitakyushu, Japan; j Kyushu Rosai Hospital, Kitakyushu, Japan Acute disseminated encephalomyelitis (ADEM) has recently been studied in several countries due to the development and wide spread of imaging technology, but few epidemiologic studies of ADEM have been undertaken, especially in Asian countries. We conducted a multicentric study on childhood ADEM, acute transverse myelitis (ATM) and multiple sclerosis (MS) by epidemiologic approach from 1998 to 2003 at Fukuoka Prefecture in southern Japan, where almost 5 million people lived. We identified 38 patients with disseminated demyelination of the central nervous system; 26 with ADEM, 4 with TM and 8 with MS. An estimated incidence of ADEM was 0.64/100,000, median age at onset was 5.7 years, and male-female ratio was 2.3:1. The median age at onset (9.3 years) of MS was significantly higher than that of ADEM. Nineteen patients with ADEM (73%) experienced febrile illnesses within one month before the onset. As a prodromal symptom of ADEM, fever was observed in 73% of patients. Laboratory studies revealed pleocytosis (81%) and protein elevation (34%) in cerebrospinal fluid. Compared with previous reports of ADEM in other countries, the onset age and sex ratio in Japan were similar, but the proportions of fever and seizures were higher. There might be distinct clinical features of ADEM in Asian countries. PP16-01 Intraneural γδ T cells during experimental autoimmune neuritis T. Fujioka a , T. Kudeken a , T. Kiyozuka a , Y. Iwasaki, T. Kurihara and A. M. Rostami b a Toho University, Tokyo, Japan; b Thomas Jefferson University, Philadelphia, USATo investigate the infiltration of γδ T cells in peripheral nervous system during autoimmune neuritis, FACS analysis of the infiltrating lymphocytes and quantitative PCR for the intraneural expression of γδ TCR mRNA in the cauda equina (CE) of female Lewis rats with SP26induced experimental autoimmune neuritis (EAN) were sequentially studied. Total RNA was extracted from CE sampled at four, seven, 10, 14, 18, 21, 26 and 32 days post-immunization (DPI; n = 4). Complementary DNA was then analyzed by quantitative PCR for γδ TCR and house keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expressions. The value of γδ TCR mRNA products was expressed by the ratio to the value of GAPDH PCR products of the same rat. For FACS analysis, mononuclear cells recovered from the homogenated CE sampled at 13 and 17 DPI (n = 6) were stained by anti-rat CD3-FITC or γδ TCR-PE monoclonal antibody. The rats developed EAN at 10 DPI, peaked at 14 to 18 DPI then recovered. γδ TCR mRNA was negligible at 0 DPI, increased at 4 DPI only transiently then started to increase at 10 DPI again. It reached the peak at 14-18 DPI then recovered to the basal level by 32 DPI. FACS analysis revealed that γδ T cell populations among total T cells in the CE were always higher than those in the other organs (peripheral blood, lymph nodes and spleens) of the same rats. Our data suggest two waves of γδ T cell infiltration one of which might be proinflammatory while the other might be anti-inflammatory. Tumor necrosis factor-alpha (TNF-α) and inducible nitric oxide synthase (iNOS) are involved in inflammatory processes such as Guillain Barré Syndrome and its animal model, Experimental Autoimmune Neuritis (EAN). We have investigated the presence of TNF-α and iNOS in peripheral nervous system during EAN. At different stages of the disease, spinal roots adjacent to lumbar intumescence, cauda equina, sensitive ganglia and sciatic nerves were obtained from induced-EAN Lewis rats and processed for double iNOS and TNF-α fluorescence immunohistochemistry. Large oval TNF-α + cells and smaller rounded TNF-α + /iNOS + cells were evidenced. All experimental groups, including the control group showed TNF-α + cells around ganglia and in perineurium, with slight differences among groups. At the onset of clinical signs, few TNFα + /iNOS + cells were found in blood vessels of cauda equina and sciatic nerve. During the acute phase there has been an increase in the amount of these cells around vessels and inflammatory infiltrates in ganglia, cauda equina and the other spinal roots. Just before the peak stage, TNF-α + /iNOS + cells in these regions and in sciatic nerve were widely distributed around numerous infiltrates. In the recovery stage, some TNF-α + /iNOS + cells have been still found in cauda equina and sciatic nerve vessels, while in the resolution stage they were not evidenced. Evolution of EAN clinical signs may be related to TNF-α and iNOS-immunoreactive cells in different nervous regions. Probably these cells are stimulated to produce TNF-α and iNOS simultaneously before reaching peripheral nervous microenvironment. PP16-03 Expression of caveolin-1 in experimental autoimmune neuritis The expression of caveolin-1 and the related molecule endothelial nitric oxide synthase (eNOS) was analyzed in the sciatic nerves of Lewis rats with experimental autoimmune neuritis (EAN). Western blot analysis showed that caveolin-1 was significantly increased in the sciatic nerves with EAN upon initiation of cell infiltration during the early and peak stage (days 10 and 14 post-immunization, p.i. ), and declined thereafter. The pattern of eNOS expression over the course of EAN largely matched that of caveolin-1. Immunohistochemistry showed that, in EAN lesions, intense caveolin-1 immunostaining was seen in ED1-positive macrophages, as well as intensely in vascular endothelial cells and Schwann cells, which constitutively expressed caveolin-1 in normal sciatic nerves. Consequently, we postulated that caveolin-1 expression increased in the sciatic nerves with EAN, possibly mediating either molecular trafficking and nitric oxide generation partly through the activation of eNOS in vascular endothelial cells and Schwann cells, as well as in inflammatory macrophages in EAN and /or cellular apoptosis of inflammatory cells. PP16-04 Regulation of genes for immune system-associated molecules in Schwann cells (SC) by cyclic adenosine monophosphate (cAMP) in vitro RP Lisak, B Bealmear, JA Benjamins, B Yao and S Land Department of Neurology, Wayne State University, Detroit, USA; Center for Molecular Medicine and Genetics, Wayne State University, Detroit, USA As part of our ongoing studies of interactions between cytokines and SC, we studied the effects of cAMP on gene regulation at early (6 h) and intermediate (24 h) time points. Neonatal rat SC were incubated with l mM 8-Br-cAMP for 6 and 24 h. RNA was extracted from SC and analyzed with Affymetrix microarray gene chips. Up or down regulation of expression compared to unstimulated SC was set at very high stringency (p < 0.001). We found changes in expression of many genes including several originally associated with the immune system. At 6 h we observed upregulation for genes for tumor necrosis factor (TNF)-induced protein 6, CD200, cytokineinduced neutrophil chemoattractant-2, CD24, CXCL2 and transforming growth factor (TGF)B-induced transcript 4. At 24 h there was upregulation of the genes for CXCR4, integrinB4, hypoxia induced factor (HIF)-la responsive RTP801, HIF-la and TGFB-induced transcript 4. Down regulation was noted for genes for IgG Fc binding protein 1, TNFRSF member 12a (receptor for TWEAK), integrinB, integrinB-binding protein 1 and endothelial monocyte-activating polypeptide. Thus SC differentiation is associated with changes in expression of genes for immune system associated molecules, which may be important in SC differentiation, and in interaction of SC with the immune system. and 6%): Anti-GQ1b IgG antibodies were highly positive (81 and 62%): MRI showed brain abnormality (3 and 23%): Abnormal EEG findings were noted (25 and 57%). Brain abnormality was detected in FS, although the frequency was lower than in BBE. PP16-13 A bedside prognostic scoring system for Guillain-Barré syndrome R. van Koningsveld a , E. W. Steyerberg b , P. A. van Doorn c , B. C. Jacobs a,c Departments of a Neurology, b Public Health, c Immunology; Erasmus Medical Center, Rotterdam, The NetherlandsObjectives. Guillain-Barré syndrome (GBS) is an immune-mediated polyneuropathy with a highly variable clinical course and outcome. In this study a bedside scoring system was developed and validated to predict in an early disease phase the long term prognosis, i.e. The derivation set included 388 GBS patients from two Dutch trials and one Dutch pilot study. Sixteen potential prognostic factors were tested univariately. A scoring system was developed based on the regression coefficients of these variables in a multivariable logistic regression model. Model performance was quantified regarding discrimination (area under receiver operating curve (ROC)) and calibration (graphically). Internal validation was performed by bootstrap resampling.Results. Several factors had a significant prognostic value in univariate analysis, but the 3 key predictive variables in multivariate analysis were age, preceding diarrhoea and GBS disability grade at two weeks after admission. The scoring system ranged from 0 to 7, including three categories for age (≤40, 40-60, >60), two for diarrhoea (absent/present) and five for GBS disability grade at two weeks. The predictions showed a high association with observed outcomes and very good discriminative ability (area under ROC 0.85). The scoring system is currently validated in another large set of GBS patients.Conclusion. The scoring system based on three simple bedside clinical characteristics present at two weeks after admission accurately predicts the inability to walk independently at six months in patients with GBS.In axonal Guillain-Barré syndrome, anti-ganglioside antibodies could cause peripheral motor nerve conduction failure. The earliest pathological change is lengthening of the nodes of Ranvier in the ventral roots. Saltatory conduction along the myelinated axons depends on the accumulation of voltage-gated sodium (Nav) channels at nodes. Paranodal axo-glial junctions flanking nodes are important for the appropriate ion channel clustering. Nav channel dysfunction may cause the initial motor nerve conduction block in axonal Guillain-Barré syndrome. By immunizing with gangliosides, rabbits produced anti-GM1 IgG antibodies and developed acute limb weakness with a monophasic course. Electron micrographs showed abnormally lengthened nodes in ventral roots from the paralyzed rabbits. Immunohistochemical studies revealed deposit of IgG, C3 component of complement, and membrane attack complex on the nodes in ventral roots from the rabbits during the acute phase of the illness. The number of affected nodes with complement deposit was reduced during the recovery phase. Clusters of Nav channels, βIV spectrin (a cytoskeletal protein at nodes), and contactin-associated protein (Caspr; axonal component of the paranodal junction) were altered or disappeared on the abnormally lengthened nodes with complement deposit. Anti-GM1 antibodies mediate complement-dependent destruction of nodes of Ranvier and disrupt Nav channel clusters. These findings confirm the hypothesis that Nav channel dysfunction is responsible for the initial nerve conduction block and consequent motor weakness in axonal Guillain-Barré syndrome. PP16-17 Higher sensitivity in detecting anti-myelin associated glycoprotein (MAG) IgM autoantibody when using antigen purified from human brain Materials and methods: Seventy-five sera from patients suspected of having autoimmune neuropathy, and sent to Focus Diagnostics for MAG IgM testing (Western blot; human MAG), were split, de-identified and sent blind to ARUP Laboratories. These 75 sera were assessed for the following IgM autoantibodies at ARUP Laboratories: anti-myelin by indirect fluorescent antibody (IFA) using primate peripheral nerve; anti-MAG by Western blot using antigen purified from primate; anti-MAG by enzyme immunoassay (EIA) using antigen purified from human brain; anti-sulfate-3glucuronyl paragloboside (SGPG) by EIA using antigen purified from bovine.Results: When compared to the MAG IgM results obtained by Focus Diagnostics (Western blot using human MAG), the following percent agreement, sensitivity and specificity were calculated for the MAG IgM Western blot (primate MAG) and MAG IgM EIA (human MAG): 85. There were 11 of 40 MAG Western blot (human MAG) positive sera that were negative by MAG Western blot using antigen purified from primate. Nine of these 11 (81.8%) sera were also positive by MAG EIA and myelin IFA. There were 7 of 35 MAG Western blot (human MAG) negative sera that were positive by MAG EIA. Six of these 7 (85.7%) sera also gave positive results for SGPG by EIA.Conclusion: When assessing sera from patients suspected of having autoimmune neuropathy for MAG IgM, these data suggest a higher sensitivity in methods using human MAG. Moreover, these data suggest that EIA may be more sensitive than Western blot in detecting IgM autoantibody against human MAG. PP16-18 Epidemiology of chronic inflammatory demyelinating polyneuropathy in Japan Chronic inflammatory demyelinating polyneuropathy (CIDP) is characterized as an acquired and symmetrical motor and sensory disturbance brought about by immune-mediated segmental demyelination in peripheral nerves. We aimed to clarify the crucial prevalence and incidence rate in Japan and performed a large epidemiologic study throughout Japan. This study covered the whole Japanese population and investigated the number of CIDP patients in combination with each sex, age group (adult and juvenile) and geographic distribution. We investigated the prevalence and incidence number of patients in general and university hospitals with either a department of neurology, internal medicine or pediatrics during November 2004 to August 2005. The number of patients was reported by all hospitals with a department of neurology and by randomly extracted hospitals with internal medicine and pediatrics corresponding to its number of beds. The number of Japanese patients used for the calculation of the prevalence and incidence rate follows the national census of 2005. As a result, we clarify that the annual prevalence rate is 1.91/100,000 and the incidence rate is 0.57/100,000 in Japan, which is very similar to the results in other counties. In conclusion, the epidemiology of CIDP shows universality not only from the aspect of ethnic distribution but also geographical distribution worldwide as well as in Japan. Chronic Inflammatory Demyelinating Polyneuropathy (CIDP) is an autoimmune disease that targets the myelin sheaths in peripheral nerve. The target antigen is unknown and there is no linkage to the major histocompatibility (MHC) locus that is involved in protein antigen presentation. As glycolipid antigens are implicated in other autoimmune neuropathies, we investigated CIDP patients for DNA polymorphisms in CD1A and CD1E, members of the CD1 gene family involved in the processing and presentation of lipid antigens to T-cells. Patients with IgM monoclonal gammopathy (MG) associated neuropathy were also investigated. We found that patients with CIDP (n = 23) were more than twice as likely to have the CD1E 01/01 genotype than normal controls (n = 28) (p < 0.01). A negative association between MG neuropathy (n = 15) and CD1E 01/01 was also observed, but this finding did not reach statistical significance (p = 0.018). The association with CD1E 01/01 implicates immune reactivity to a lipid antigen in the pathogenesis of CIDP. Furthermore, the gene product of CD1E 01 may have a higher affinity for a putative CIDP antigen than other forms of CD1E, or homozygosity may result in higher expression of 01, facilitating antigen presentation, and enhancing susceptibility to disease. This points to the CD1E pathway as a potential target for therapeutics. PP16-20 Diagnostic criteria and nationwide survey of Crow-Fukase syndrome in Japan We previously reported that VEGF was extremely elevated in Crow-Fukase syndrome (CFS) and was a key molecule of the pathogenesis of CFS. According to our clinical and experimental studies, we proposed the diagnostic criteria of CFS. To confirm the usefulness of the diagnostic criteria, we conducted a Japanese nationwide survey of CFS in 2003. Furthermore, we investigated the efficacy of various treatments for CFS. The estimated number of patients was about 340. Detailed medical questionnaires of 84 CFS patients were obtained (males: 49, females: 35, mean age: 57 years old). However, mean annual frequency of hospitalization (2.7 times) and absence of any patient showing complete recovery indicated that CFS is still an intractable disease. Polyneuropathy (100%), edema (97%), and skin lesions (95%) were the most frequent manifestations, followed by organomegaly (79%) and endocrine abnormalities (78%). On laboratory examination, M-protein (89%) and high serum VEGF (98%) were detected in these patients. These findings surprisingly resembled the previous Japanese nationwide survey conducted by Nakanishi in 1982, but differed from the report of Dispensieri et al. According to Dispenzieri's diagnostic criteria, 11% of patients were not correctly diagnosed. Our proposed diagnostic criteria, which includes serum VEGF, correctly diagnosed CFS in all patients. Treatment by peripheral blood stem cell transplantation with high dose chemotherapy was increased and most responded well compared to conventional therapy. PP16-21 Highly concentrated vascular endothelial growth factor (VEGF) in platelets in Crow-Fukase (POEMS) syndrome We report a marked difference in concentration of vascular endothelial growth factor (VEGF) between serum and plasma in patients with Crow-Fukase syndrome (CFS). The serum/plasma VEGF levels in four CFS patients were 8634/152, 5203/176, 3724/127 and 868/13 pg/ml, respectively. It is predicted that in CFS, local VEGF concentration is markedly elevated by aggregation of platelets containing excessive VEGF and their adhesion to vascular walls, resulting in excessive physiological activities of VEGF, which are considered deeply involved in the characteristic clinical manifestations observed in CFS. Our findings provide important information for developing more effective therapeutic trials. PP16-22 Peripheral neuropathy in patients with multiple sclerosis Background: Multiple sclerosis (MS) is characterized by the demyelination in the central nervous system, but a few studies have demonstrated the involvement of peripheral nerves. The prevalence of peripheral neuropathy in MS remains controversial. The aim of this study is to perform an electrophysiological evaluation of the peripheral nervous system in patients with MS. Methods: Subjects were 28 patients with MS, 7 males and 21 females and aged between 21 and 51 (mean 34). Motor conduction study in median, ulnar, peroneal and tibial nerves as well as sensory conduction in median, ulnar, and sural nerves were evaluated. Results: There was electrophysiological evidence of peripheral nervous system lesions in at least one nerve in 9 patients. Low sensory nerve action potential was noted in 1 patient. Conclusions: This study demonstrated the peripheral nerve involvement in patients with MS. Further investigations are needed to clarify the immunological background affecting both central nervous system and peripheral nerves. Methods: Consecutive 59 patients with MS and 61 patients with CIDP were studied. Results: Clinical evident association of the two disorders was found in the two patients (3% of the MS patients and 3% of the CIDP patients). Nerve conduction studies showed demyelination in the intermediate nerve segments without involvement of the distal nerve terminals. Another MS patient presented subclinical nerve conduction abnormalities suggestive of intermediate demyelination. CIDP associated with MS is characterized by multifocal demyelination in the nerve trunk and no involvement of the distal nerve terminals. Myelin components common to the central and peripheral nervous systems may be target molecules in such cases. PP16-24 Clinical features of the neuropathy with high titer IgM antibody against gangliosides with disialosyl residue IgM paraproteins that bind to gangliosides with disialosyl residue have been reported to be specifically associated with sensory ataxic neuropathy. Among the sera in which we have examined antiganglioside antibody titer, there are 5 sera with high IgM antibody titer against GD3, GD1b, GT1b and GQ1b. We investigated the clinical feature, clinical course and response to the treatment of each case. The remaining one patient, who had IgM with high titier against GD3, GT1b and GQ1b but with very low titer against GD1b, had sensori-motor neuropathy with no ataxia. Patients were treated either by intravenous immunoglobulin (IVIg), corticosteroids, plasma exchange, immunoadsorption or immunosuppressants. Two patients had IgM M-protein, and the other 2 patients had malignant hematological diseases. The treatment to the hematological diseases in the latter 2 patients also improved neuropathy. Several treatments were effective but the effects were temporary. PP16-25 Therapeutic effects of single administration of Cyclophosphamide in rat experimental autoimmune neuritis, a preclinical model of autoimmune peripheral neuropathies High dose immunosuppressive therapies have been used to treat autoimmune diseases, including autoimmune peripheral neuropathies. These agents have been used to treat some cases of Guillain-Barré syndrome (GBS), Chronic Inflammatory Demyelinating Polyradiculoneuropathy (CIDP) and multifocal motor neuropathy (MMN), resistant to usual treatments, such as plasma exchange (PE) and intravenous immunoglobulin (IVIg). The long term use of high doses of these immunosuppressants is however limited due to their severe side effects: bone marrow suppression, hair loss, upset stomach and development of hemorrhagic cystitis. In order to assess the beneficial effect of short term treatment with lower doses of the alkylating immunosuppressive drug Cyclophosphamide (CY) on the development of a preclinical model of GBS and CIDP, the experimental autoimmune neuritis (EAN) in Lewis rats, a single dose of CY was administered to the animals 14 days post-immunization. The data obtained in our experiment shows that a single low dose administration of CY was highly effective in preventing clinical signs of EAN. These data suggest that CY deserves further consideration for its use in the treatment of both acute and chronic autoimmune peripheral neuropathies. In particular, since the acute phase of human GBS is usually ranging between 2 and 4 weeks, it is possible that one single administration of the drug would be sufficient to influence the course of the disease, thus avoiding the well known side and toxic effects provoked by prolonged exposure to CY. PP16-26 Intravenous immunoglobulin therapy for Miller Fisher syndrome Background: There is no evidence whether intravenous immunoglobulin (IVIg) has beneficial effects for Miller Fisher syndrome (MFS) patients.Methods: Clinical recovery was reviewed in a consecutive series of 92 patients with MFS. The time-course of recovery was compared in patients treated with IVIg (n = 28), those treated with plasmapheresis (PP; n = 23), and those who did not receive these immune modulating treatments (n = 41).Results: The times required to start amelioration of external ophthalmoplegia and ataxia were slightly shorter for the IVIg group (ophthalmoplegia; median, 12.0 days; p = 0.04, ataxia; median 8.0 days, p = 0.027) than the control (ophthalmoplegia; 13.5 days, ataxia 10.0 days) groups, but the times required to disappear external ophthalmoplegia and ataxia were similar between the IVIg and the control and between the PP group and the control. One year after onset, 89 (96%) of the 92 patients had no ophthalmoparesis and ataxia irrespective of receiving immunomodulating treatment.Conclusions: In MFS, IVIg slightly hastens the amelioration of ophthalmoplegia, but does not affect the outcome, presumably because of a good natural recovery. Variant type Guillain-Barré syndrome (V-GBS) patient's serum has miniature endplate potential (MEPP) frequency increase (MFI; so-called α-latrotoxin like effect) with anti-ganglioside antibodies, and these correlate to clinical symptoms. Mechanism of MFI is speculated as complement dependent nerve terminal destruction by autoantibody. The autoantibody and complement make membrane attack complex and create a lot of pores on the nerve terminal membrane. Thereafter, too much Ca + concentration gain injures the nerve terminal, resulting in disappearance of MEPP at the end. We were able to confirm this process on the mouse diaphragm, and we tried immunoglobulin (Ig) and prednisolone (PSL) treatment to this in vitro model. We chose a V-GBS patient whose serum had high MFI and anti-ganglioside antibodies. We prepared a C 57 BL/6J mouse diaphragm preparation bathed in the patient's serum (0.5 cc) and recorded MEPP using conventional grass microelectrode technique. We added immunoglobulin (Ig), prednisolone (PSL) or both of them to the patient's serum. There was complete suppression of MFI when we used 10 mg/ml Ig or 1 mM PSL. MEPP frequency increase was not suppressed by 0.1 mM PSL, but the appearance of the MEPP lost fibers was suppressed. One mg/ml Ig and 0.01 mM PSL did not influence the patient's serum action. To confirm the synergistic effect of Ig and PSL, we used patient's serum containing the 0.1 mM PSL and 2 or 5 mg/ml Ig. However, there was no complete MFI suppression. We could not confirm usefulness of the Ig and PSL combination therapy by this in vitro model. 1 Kitasato University, Sagamihara, Japan; 2 The University of Tokyo, Tokyo, Japan; 3 Institute of Animal Reproduction, Kasumigaura, Japan; 4 Japan Red Cross Hospial, Tokyo, Japan; 5 Nationall Hospital Organization, Nagoya Med Center, Nagoya, JapanWe have previously reported that thymic myoid cells are possibly stemmed from plural sources and that some myoid cell clones produce growth factors, including an 80-kDa haemopoietic factor (80-K) and 100-kDa haemopoietic biglycan (100-K), besides AChR. Those factor-producing cells were also abundantly detected in the hyperplastic myasthenic thymi (H-MG-thymi). AChR (+) cells and myogenin (+) cells were in a subset of them, suggesting that 100-K (+)/AChR (+) double positive cells are matured myoid cells, whereas 100-K (+)/AChR (−) single positive cells their precursors. Numeral increases of myoid cells occurred at precursor stages, followed by concurrent production of large amounts of AChR and growth factors that may affect the AChR specific breakage of the anergic state. 80-K/100-K appear to be a useful hallmark for understanding the immunological state of H-MG-thymi. However, little is known about the mechanisms of development of myoid cells that produce those factors, although recent studies on skeletal muscle development suggested that biglycan, decorin and other factors such as Pax3/7 are differentially expressed in embryonic and fetal myoblasts. In the present report, we use different types of myoid cells cloned from mice and rats, and hyperplastic thymic tissues to correlate between the regulatory factors and the growth factor production. We are currently testing the role of regulatory factors of myoid cells and their immunostimulatory products for better understanding of MG. Heading: The aim of this study is to investigate the pathogenesis and characteristics of muscle-specific tyrosine kinase (MuSK) antibody in experimental autoimmune myasthenia gravis (EAMG) rat.Background: Some of generalized seronegative MG patients have antibodies (Ab) against MuSK. We investigated the pathogenesis of MuSK Ab using rat.Methods: Female Lewis rats, aged 8 weeks were inoculated twice in multiple intradermal sites, either MuSK protein (10-100 μg) in complete Freund's adjuvant (CFA) and Bordetella pertussis as co-adjuvant or the controls. Soluble mouse MuSK-6xHis protein was purified from HEK293.Results: The weights of MuSK-immunized rats were lower than those of the control rats. The titers of antibodies to MuSK raised markedly compared with those of the controls. The electrophysiological examination using diaphragm was negative. The quantity of AChR and MuSK at the endplates of limb muscles were reduced in MuSK-immunized rats, compared with those of the control rats. The morphological changes, AChR-declustering muscle fibers have appeared in the MuSK-immunized rat (EDL, white muscle > soleus, red muscle).Conclusions: Our results suggest that anti-MuSK antibodies in MuSKimmunized rat may down-regulate MuSK and AChR, lead to the morphological changes in motor endplates. Further investigations will draw the conclusion whether MuSK Ab may be pathogenic or not. PP17-03 The thymic theme of acetylcholinesterase splice variants in myasthenia gravis Adi Gilboa-Geffen 1 , Paul Lacoste 2 , Lilach Soreq 1 , Geraldine Clairac 2 , Frederique Truffault 2 , Iftach Shaked 1 , Hermona Soreq 1,3 and Sonia Berrih-Aknin 2 1 The Hebrew University of Jerusalem, Israel; 2 CNRS UMR8078-CCML, LePlessis Robinson, Paris, France Myasthenia gravis (MG) is an autoimmune disease mediated by antibodies towards nicotinic acetylcholine receptors. Thymic abnormalities such as hyperplasia and thymoma are found in most MG patients. Here, we report that modified acetylcholinesterase (AChE) gene expression and properties are involved in these thymic pathologies. Microarray analysis demonstrated attenuation of the general decrease in gene expression, which is characteristic of adult human thymus, in patients with MG-induced thymic hyperplasia. This was accompanied by reduced expression of DNA replication-associated transcripts and by reduced levels of the "synaptic" AChE-S mRNA. Thymic extracts from MG patients further presented elevated amounts but reduced hydrolytic activities of the MG-induced AChE-R variant as compared with adult controls, accompanied by translocation of both AChE-R and the signaling protein kinase PKC-βII from the cytoplasmic to the membrane-associated fraction. To explore possible causal association of the modified AChE splicing pattern with thymic composition and function, we employed up-regulation of specific thymic AChE variants TgR mice overexpressing human (h)AChE-R and TgS mice overexpressing the membrane-associated (h)AChE-S variant. Both showed smaller thymic medulla than strain-matched FVB/N controls, suggesting differentiation imbalances. Intriguingly, TgS mice displayed smaller thymus weight, CD3 + -high mature cell fractions with higher rate of spontaneous apoptosis as compared with controls. In contrast, TgR mice showed heavier thymuses, selectively enlarged CD4 + CD8 + fractions and protection of cultured thymocytes from apoptosis. Together, the overexpression of AChE-R variant could explain the higher number of thymocytes in both the transgenic model and the human thymus associated with myasthenia gravis. PP17-04 The thymus is a source of B-cell survival factors -APRIL and BAFFin myasthenia gravis Mathula Thangarajh 1,2 , Thomas Masterman 1 , Lars Helgeland 3 , Uroš Rot 4 , Malin V. Jonsson 5 , Geir Egil Eide 6 , Ritva Pirskanen 7 , Roland Jonsson 2 , Jan Hillert 1 ml). Anti-MuSK (−) subgroup had also higher IFN-γ levels compared to anti-MuSK (+) subgroup when stimulated with AChR (2.4 vs. 0.0 pg/ml, p = 0.03). IL-13 and IL-10 secretion levels and proliferative responses were not different between disease subgroups. Although regulatory cytokine activity is detected in MG spontaneously, differences between disease subgroups according to Ab are implicated by these results. This study was supported by the Brain Research Society (BAD) and Istanbul University Research Fund. PP17-08 Autoreactive T cells mediate NK cell degeneration in autoimmune disease Mechanisms underlying this NK cell degeneration are not known. Here we show that, in an experimental model of human autoimmune myasthenia gravis induced by a self-antigen, the acetylcholine receptor, NK cells undergo expansion during the initiation of autoimmunity, followed by significant degeneration concomitant with the establishment of the autoreactive T cell response. NK cell degeneration was mediated by interleukin-21 derived from autoreactive CD4 + T cells. NK cell degeneration may signify the transition from innate immune triggering to emergence of autoreactive T cells, serving as a means evolved by the immune system to control excessive autoimmunity. PP17-09 Comparative analysis of two different assays in the detection of anti-MuSK antibodies M. Marino a , F. Scuderi a , C. Provenzano a , A. Evoli b and E. Bartoccioni a . a Institute of General Pathology and b Department of Neurosciences, Catholic University, Rome, ItalyAnti-MuSK autoantibodies have been associated to "seronegative Myasthenia Gravis" (SNMG). Until now these antibodies have been detected by means of two different techniques: a home-made immunoblotting of biotinylated proteins assay (IBBA) and a radioimmunoprecipitation assay (IPA). The IBBA uses biotin-labeled membrane proteins from TE671 cells while the IPA uses 125-I-labeled purified recombinant antigen, the results being given as nmoles 125-I-MuSK precipitated per liter of serum. We compared home made IBBA with commercial IPA (RSR Limited). testing 130 sera: 20 from healthy blood donors, 65 from SNMG patients, and 45.from other neurological pathologies. No anti-MuSK abs were detected in control sera by both assays, while they were detected in 38 out of 65 (58.5%) SNMG patients tested by IPA, and in 42 out of 65 (64.6%) tested by IBBA.Five out of 65 sera tested were IBBA(+) and IPA(−), while only one serum was IBBA(−) and IPA(+); the concordance between the two tests was 95.4%, with comparable values of sensitivity and specificity. IBBA is a qualitative test, which detects several immunoreactivities against native muscle antigens; it is complex, not easy to standardize for routine and requires the support of cell culture facilities. IPA is a quantitative test, which employs a recombinant antigen (extracellular fragment, 1-490 aa) and allows to titre the anti-MuSK antibodies in patient sera, however, it requires the support of radioisotopic facilities. PP17-10 A new sandwich ELISA method to detect antibodies against native form of ryanodine receptor The purpose of this study is to design a new measurement method to detect serum autoantibodies against the ryanodine receptor (RyR) of striate muscle. With this new method of sandwich ELISA using the complex of anti-RyR monoclonal antibody and native form of RyR from rabbit striate muscle as the target antigen, we measured titers of the autoantibody to RyR from serum samples of 80 patients with myasthenia gravis (MG), 34 disease controls and 23 normal subjects. When titers that exceeded the average + 3 × standard deviation of normal samples was defined as the positive, 31 patients with MG (39%) turned to be positive. On the other hand, all samples from the disease and normal controls were negative. Furthermore, it tended to be high in female and in patients with the generalized MG. In spite of no detection of antibodies to RyR from the samples without antibodies to acetylcholine receptor, there was no correlation with these two autoantibodies. These results suggest that our new method is useful to detect anti-RyR antibodies and is expected as the method to recognize the pathophysiological condition of each myasthenic patient with this antibody. PP17-11 Elevation of IL-12 p40 and its antibody is a specific serum marker for myasthenia gravis with thymoma We measured the serum levels of various cytokines, and their antibodies in patients with myasthenia gravis (MG). The levels of IL-12 p40 were significantly elevated in 53.3% of MG patients with thymoma (n = 15). Titers of anti-IL-12 p40 antibodies were significantly elevated in 40.0% of MG with thymoma. There was a significant correlation between IL-12 p40 and its autoantibodies in thymoma with MG; however, there was no correlation in MG with LFH, MG with normal thymus or thymoma without MG. The titers of IL-12 p40 did not have a significant correlation with the histopathology of thymoma according to the WHO classification. Furthermore, the levels of IL-12 p40, anti-IL-12 p40 antibodies and anti-IL-12 p70 antibodies did not correlate with serum titers of anti-acetylcholine receptor (AChR) antibodies, clinical severity, or ratio of 201 Tl accumulation in the thymus. These observations suggest that both IL-12 p40 and its autoantibodies are associated with the immunopathology of MG with thymoma, which has distinct immunopathology compared to other MG. PP17-12 Long term effect of infrasternal mediastinoscopic thymectomy in myasthenia gravis Background: Endoscopic thymectomy is now widely performed because of relatively low invasion and less stress for patients. However its long term effect has not been sufficiently evaluated. Objective: To assess the long term effect of infrasternal mediastinoscopic thymectomy (IMT) and compare with transsternal thymectomy (TT) in myasthenia gravis (MG) patients. Methods: Among 41 MG patients who underwent thymectomy in our institute between January 1997 and December 2000, 16 patients who received IMT and 17 who received TT were enrolled in this study. QMG score and antiacetylcholine receptor antibody (anti-AChR) titer were evaluated before and 5 years after the surgery. Intracellular cytokine of peripheral blood CD4 − positive T cells were analyzed by flowcytometry in 9 patients who underwent IMT, and IFNγ/IL-4 ratio was calculated. Results: After 5 years, QMG score reduced from 6.9 to 1.9 (p < 0.01) in IMT group and 7.5 to 2.5 (p < 0.01) in TT group. Anti-AChR titer reduced from 68.6 to 40.0 (p = 0.048) in IMT group and 201.1 to 106.2 (p = 0.017) in TT group. Conclusion: IMT is as effective as TT, and it is considered to be suitable for MG. IMT seemed to alleviate MG symptoms by correcting the Th2 shift. PP17-13 Effect of thymectomy for late onset non-thymomatous myasthenia gravis N. Kawaguchi a , S. Kuwabara a , Y. Nemoto a , H. Takahashi a , T. Fukutake a , K. Arimura b , M. Osame b , and T. Hattori a a Department of Neurology, Chiba University, Graduate School of Medicine, Chiba, Japan; b The Third Department of Internal Medicine, Kagoshima University, Kagoshima, JapanObjectives: To investigate whether thymectomy is beneficial for nonthymomatous myasthenia gravis patients with mild generalized weakness and onset age >50 years.Patients and methods: Clinical course and outcomes were compared between 10 patients receiving thymectomy and 12 without it.Results: At the end of follow-up (mean 9.6 years after onset), the thymectomized group showed the greater percentage of clinical remission (no symptoms with or without medication; 50% versus 17%; p = 0.11), and the less frequency of the presence of generalized symptoms (30% versus 75%; p < 0.05).Conclusions: Thymectomy appears to be effective for late onset, nonthymomatous patients with generalized myasthenia gravis. Objectives: To investigate whether E-C coupling of muscle is impaired in patients with generalized myasthenia gravis (MG). Methods: A total of 51 patients with MG were studied. Compound muscle action potentials (CMAPs) of the abductor pollicis brevis, and movement-related potentials using an accelerometer placed at the thumb tip were recorded after median nerve stimulation at the wrist. The E-C coupling time (ECCT) was estimated by a latency difference between CMAP and movement-related response. Antibodies against ryanodine receptor (RyR) and muscle specific receptor tyrosine kinase (MuSK), as well as acetylcholine receptor, were measured by immunoassays. Results: Compared with age-matched normal controls (n = 31), the mean ECCT was longer in patients with MG (p = 0.01). ECCT had no significant correlation with clinical severity, disease duration, and anti-RyR or MuSK antibodies, whereas the mean ECCT was shorter for patients treated with FK506 than for those not receiving this treatment (p = 0.03). PP17-15 Tacrolimus treatment in elderly patients with myasthenia gravis H. Takahashi, N. Kawaguchi, Y. Nemoto, T. Hattori Department of Neurology, Graduate School of Medicine, Chiba University, Chiba, Japan <Background> Tacrolimus is an immunomodulating agent being widely used for patients with myasthenia gravis (MG), rheumatoid arthritis, and organ transplantation in Japan. Tacrolimus have effect to inhibit antiacetylcholine receptor (AChR) antibody production through suppressing helper B cell, and help uptake corticosteroid into nucleus. We evaluate the effect and adverse effect of tacrolimus for elderly patients with MG. <Object and method> Two groups are evaluated with myasthenia gravis activities of daily living (MGADL) score, and dose of corticosteroid used in comparison before initiation and 12 months after. One group consists of 14 corticosteroid dependent patients (M:F = 9:5, 52-76 year-old); the other consists of 12 patients treated without corticosteroid (M:F = 5:7, 51-82 year-old). <Result> In corticosteroid dependent group, 78.5% of patients presented clinical improvement 12 months after initiation of tacrolimus. 57.1% of this group could reduce the amount of daily corticosteroid. The average dose of corticosteroid decreased from 28.9 mg every other day to 16.9 mg every other day. In group treated without corticosteroid, 67% of patient presented clinical improvement 12 months after initiation of tacrolimus. Their average MGADL score decreased from 5.50 points to 2.58 points. The average number of Anti-AChR antibody decreased from 56.0 to 23.5. 15% of all patients showed deterioration of glucose tolerance. <Conclusion> Even for elderly MG patients, tacrolimus could be a useful choice of treatment. PP17-16 Tacrolimus as a treatment for severe myasthenia gravis Tacrolimus is a macrolide immunosuppressant that is widely used in liver transplantation. Tacrolimus is authorized for treatment of myasthenia gravis (MG) in Japan, and can result in a reduced requirement for steroids. We have used tacrolimus for 12 patients with severe MG who had experienced an MG-related crisis episode, and observed these patients for more than two years. Just after the critical attack, the patients were treated using plasmapheresis, then with methylprednisolone(mPSL) pulse therapy, and tacrolimus was started during gradual tapering of PSL. Four of the 12 patients received this course of treatment as their initial therapy, whereas the other eight patients had previously undergone treatment without tacrolimus, but had experienced crisis episodes several years after treatment. Five control patients were treated with immunosuppressants other than tacrolimus, such as azathioprine (AZP) or mizoribine; two of these patients were initially treated with AZP but were then changed to tacrolimus after the crisis. Patient response was evaluated based on the MG-ADL scale, the anti-acetylcholine receptor (anti-AChR) antibody titer and the serum tacrolimus level. All tacrolimus-treated patients showed an improved MG-ADL scale and required a lower corticosteroid dose, in parallel with an increase in serum tacrolimus concentration, however, the anti-AChR titer did not decrease significantly. These results suggest that, tacrolimus is a potent tool for treatment of MG, even in severely affected patients; tacrolimus is at least as effective as other immunosuppressants and does not show any side effects, although it failed to prevent relapse due to fatigue or infection. PP17-17 Treatment of late onset myasthenia gravis in Japan Y. Takata, T. Arai, K. Yokoyama, Y. Mizuno Department of Neurology Juntendo University School of Medicine, Japan The patients of myasthenia gravis (MG) are about 15,000 patients out of 1.2 billion populations. Recently, Japan changed drastically to a high speed of aging society, it is very likely that the number of late-onset MG (clinical onset after 60 years of age) increase. The purpose of this study was to investigate the late-onset Japanese MG patients in our hospital and compare the early-onset cases for better characteristic and therapeutics. Total of 20 late-onset MG patients were analyzed and compared to over 100 early-onset MG. The clinical outcome of thymectomy in early-and late-onset myasthenia gravis (MG) and the correlation to MG severity, treatment, and anti-Ach receptor antibody were examined. Because remission of late-onset MG after thymectomy is about 30% of the patients and is same rate as of non-thymectomized MG patients. There seemed often encountered the difficult case for making therapeutic strategy of late-onset MG because of complicating disease(s). Involuted thymus is frequently seen and the presence of muscle autoantibody did not influence the outcome of thymectomy in late-onset MG.Therefore thymectomy should always be considered shortly after MG onset in late-onset patients who showed progressive severity or Ossermann Type II. PP17-18 A new therapeutic approach for myasthenia gravis, based on the molecular recognition theory using KM mice TM S. Araga a , T. Tahara b and I. Ishida b a Fujii memorial hospital, Kurayoshi, Japan; b Kirin Brewery Co. Ltd., Yokohama, JapanMyasthenia gravis (MG) is the result of interference with neurotransmission by autoantibodies against the nicotinic acetylcholine receptor (AChR) on muscle. Based on molecular recognition theory, the complementary peptide against the epitope in MG can induce anti-idiotype antibodies (abs), and can protect against the development of experimental autoimmune MG. KM mice™ have afforded an opportunity to test this idea in human. KM mouse™ is produced by induction with a human chromosome containing Ig heavy chain and κ light chain loci using a microcell-mediated chromosome transfer technique. KM-mice™ were immunized with the complementary peptide for amino acid residues 61-76 of α-chain of the human AChR, followed by booster immunization. Sensitized spleen cells were fused with SP2/0 cells to produce monoclonal abs (mAb). We obtained four mAbs to recognize the idiotype abs in MG. Anti-complementary peptide abs using KM mice™ could provide a useful tool for treatment of MG. PP17-19 Clinical and serological study of myasthenia gravis in WuHan, China Ocular and childhood cases of myasthenia gravis (MG) are relatively more common in Oriental than in Caucasian populations, but there have been no comprehensive serological studies on patients from China. 50% of the patients were children (under 15 years), and there were few patients over 50 years. Overall, 64% of the children and 66% of the adults were positive for acetylcholine receptor (AChR) antibodies. Of the 43 patients with generalized MG without AChR antibodies, only one had MuSK antibodies (2.5%) and two had VGCC antibodies indicating probable Lambert Eaton myasthenic syndrome. Despite most patients receiving prednisone, very few obtained full clinical remission, although some improvement was evident. Moreover, many of these patients have AChR antibodies, confirming the diagnosis of MG. Comparison with Chinese populations elsewhere should provide information regarding whether genetic or environmental factors influence development of MG in these children. PP17-20 A study of the factor responsible for the exacerbation of childhood-onset myasthenia gravis by T cell receptor Vβ expression repertoire Myasthenia gravis (MG) is a T cell-regulated autoimmune disease in which a pathological autoantibody response is mounted against the acetylcholine receptor (AChR). It has been suggested that activated antigen-specific or non-specific activated T cells may caused exacerbation of MG. To identify factors responsible for this exacerbation, we examined the T cell receptor (TCR) Vβ expression repertoire in the peripheral blood of nine patients with childhood-onset MG. Four patients had the latent general type, three had the general type, and two had the ocular type of MG; six patients were examined on both the remission and the exacerbation. TCR Vβ repertoire of patients was analysed by quantitative TCRVβ − receptor amplifications and spectratyping. Expansions of TCRVβ2/5/7 + T cells from patients was observed during the exacerbation regardless of the HLA DR/DQ typing, but not during remission.These data suggested that during exacerbation antigen-nonspecific T cells are activated and that the superantigens induced by bacterial or viral infection might contribute to the exacerbation of MG. PP17-21 The prevalence of MuSK antibody positive myasthenia gravis worldwide Myasthenia gravis is usually associated with antibodies to the muscle acetylcholine receptor (AChR) but in a proportion of patients with generalised myasthenia without AChR antibodies, there are antibodies instead to muscle specific kinase (MuSK). To assess the incidence of MuSK-MG systematically, we collected sera from 21 different countries in Europe and Asia, from four USA centers and from three Canadian centers. Of the 788 sera negative for AChR antibodies, 201 were positive for MuSK antibodies giving an overall positivity of 25.5%. However, the positivity varied between 0 and 49% in sera from different countries. Interestingly, within the Northern hemisphere MuSK antibody positivity peaked at 40°from the equator with the lowest positivities found in Norway (70°north) and the Philippines (15°north). Only three countries from the Southern hemisphere participated, with positivities varying between 10 and 35%. Clinical data on the MuSK antibody MG patients suggest, as previously described, that they are similar between different countries, with prominent bulbar involvement. These results, and the similarity between the incidence at equivalent latitudes in Europe, USA and Asian countries, suggest that this form of myasthenia may be associated with an environmental agent rather than with genetic predisposition. PP17-22 In vitro and in vivo characterization of subunit-specific autoantibodies from myasthenia gravis patients Muscle acetylcholine receptor (AChR) is the autoantigen in myasthenia gravis (MG). For this purpose we have successfully expressed soluble extracellular domains (ECDs) of α, β, γ and ε human AChR subunits in the yeast Pichia pastoris. These ECDs are immobilized on Sepharose beads and are used to immunoadsorb the corresponding autoantibodies from plasmas and sera of MG patients. Isolated autoantibodies against α and β subunits from four patients were tested for their ability to cause AChR internalization and degradation (antigenic modulation) in the human cell line TE671. We found that anti-α and anti-β subunit specificities were equally efficient in causing AChR antigenic modulation.We then tested the ability of MG sera and purified subunit-specific antibodies to cause experimental autoimmune MG (EAMG) in Lewis rats. Two patients' sera with high anti-rat AChR titers have been initially identified as strongly myasthenogenic when administered to the rats. The anti-α antibodies (which carried all the anti-rat AChR activity) from similar serum volumes were similarly effective in causing EAMG. In contrast the depleted from the anti-α autoantibodies serum was inefficient in causing EAMG. Similar rat cross-reactive antibody amounts against the β-subunit were not pathogenic. We are in the process to isolating large amounts of autoantibodies to various subunit ECDs from several sera (cross-reactive with rat AChR) and to further characterize them, in order to identify the characteristics of the pathogenic antibodies in MG. PP17-23 Phenotypical analysis of lymphocytes using flow cytometry in dermatomyositis with and without interstitial pneumonia Objective: Activated T lymphocytes are considered to play a key role in the pathogenesis of dermatomyositis (DM). This disease is sometimes associated with interstitial pneumonia (IP), which leads to a poor prognosis of the patients. To find out the clinical marker suggestive of associated IP in DM, we investigated intracellular cytokines and surface antigens of peripheral blood lymphocytes using flow cytometry. Methods: Thirteen patients with active DM with (n = 8, mean age 42.5 ± 15.4 yr) or without IP (n = 5, mean age 52.4 ± 24.9 yr) were enrolled in this study. Nine healthy subjects (mean age 41.3 ± 16.0 yr) were used as a control. After separation of mononuclear cells using the Ficoll-Hypaque method, we performed flow cytometry in order to investigate intracellular cytokines and surface antigens, particularly activation markers of lymphocytes. Results: Both DM patients with and without IP showed significant decreases in CD8 + CD25 + (p < 0.05), CD4 + IFN-γ + (p < 0.05) and CD4 + IL-4 + (p < 0.05) cells and a significant increase in the CD4 + IL-4 + /CD4 + IFN-γ + ratio (p < 0.05) compared with controls. There was no significant difference in these subpopulations between DM patients with and these without IP. CD8 + IFN-γ + lymphocytes were significantly decreased in DM patients with IP than those without IP (p < 0.05). CONCLUSION: Both helper and cytotoxic T cells are activated in the peripheral blood of patients with active DM. CD8 + IFN-γ + (Tc1) lymphocytes in the peripheral blood may be a key marker suggestive of associated IP in DM. PP17-24 Evaluation of serum cytokine and death receptor levels in polymyositis and dermatomyositis: Study under new criteria I. Sugimoto a , S. Kano a , T. Mikata b , S. Tsuji a and J. Shimizu a a Department of Neurology, University of Tokyo Graduate School of Medicine, Tokyo, Japan; b Department of Neurology, Shimoshizu National Hospital, Yotsukaido, Chiba, JapanBackground: It has been revealed that polymyositis (PM) is rare disease and relatively overdiagnosed with widely used Bohan's criteria, which differentiate dermatomyositis (DM) from PM only by skin changes. Recently a new criteria for PM and DM has been proposed by Dalakas based on clinical and pathological findings.Objectives: To compare the serum levels of cytokines and death receptors in patients with PM or DM diagnosed by new criteria.Methods: In 157 cases with inflammatory myopathy, 5 cases with definite PM and 14 cases with definite DM met criteria proposed by Dalakas (2003) . Serum samples of the cases obtained before treatment were examined by suspension array system (Luminex), and serum levels of GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, TNF-α, TNF-R1, TNF-R2 and DR5 were measured.Results: In DM, serum levels of IL-10, IL-6, TNF-R1, TNF-R2 and DR5 were significantly increased. In PM, although serum levels of all cytokines stayed in normal controls level, serum levels of TNF-R2 and DR5 were significantly increased.Conclusions: This is the first time approach for measuring serum cytokines and death receptors under new diagnostic criteria for PM and DM. The difference of serum cytokines profiles confirmed DM as a systemic disease and PM as a disease confined to skeletal muscle under the new criteria. Our results suggested that soluble TNF receptors may regulate TNFα mediated muscle fiber damage in both DM and PM. PP17-25 The role of autoantibodies in C-protein-induced experimental polymyositis Kuniko Kohyama, Il-Kwon Park, Mie Nakajima and Yoh Matsumoto Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, JapanIn the present study, we aimed to clarify the pathogenesis of polymyositis with regard to T cell-and B cell-mediated immunity. Immunization with partially purified skeletal myosin induced severe experimental autoimmune myositis (EAM) in Lewis rats. However, further purified myosin showed only poor myositogenisity and C-protein, a minor component of crude myosin fraction, had strong myositogenisity. To obtain a sufficient dose of the protein, we generated four recombinant C-proteins (SC1-SC4) covering the entire molecule. In addition, we synthesized twelve SC2 overlapping peptides (P1-P12) to identify epitopes for T and B cells. Immunopathological examinations revealed that the main feature of SC2induced EAM was the infiltration of T cells and macrophages and that CD8 + T cells infiltrated the muscle at the early stage. Epitope analysis demonstrated that T cells isolated from SC2-immunized rats responded to P5 and that antisera were reactive to P3, P7 and the conformational epitope(s) of the SC2 molecule. Although it was reported that pothogenic T cells were the main effector in human PM, immunization of P5 that contains T cell epitopes failed to induce EAM. On the other hand, intramuscular injection of anti-SC2 mAb induced mild but definite inflammation. These findings suggest that T cell activation is not sufficient to induce EAM and that autoantibodies play an important role in the development of EAM. We also generated recombinant cardiac C-protein fragments (CC1-CC4) and induced experimental autoimmune myocarditis (EAC) for comparison with EAM. In CC2-induced EAC, the proliferative response of T cells to CC2 overlapping peptides was not detected, whereas antibodies against various peptides were generated. These findings strongly suggest the importance of the autoantibody generation in autoimmune muscle diseases. PP17-26 The role of granulysin in muscle fiber injury of polymyositis S. Okada a , H. Nemoto b , M. Nishina a , T. Kurihara b , and T. Morishita a a Ichikawa General Hospital, Ichikawa, Japan; b Toho University, Tokyo, Japan Granulysin (GLN) is a cytolytic molecule that co-localizes with perforin in cytolytic T cells. We investigated the role of GLN in the pathogenesis of polymyositis (PM).Rat myoblast cell line L6 was cultured in RPMI 1640 (10% FCS), and differentiated to muscle fibers in RPMI 1640 (2% horse serum). Recombinant (r) GLN was co-incubated with L6 or expressed intracellularly, and GLN-mediated cytotoxicity was evaluated by MTT assay. rGLN induced cytotoxicity for L6 in a dose dependent manner when it was introduced extracellularly. Intracellular expression of rGLN also damaged L6. DNA fragmentation was observed in L6 in which rGLN was expressed, not in L6 when rGLN was administered extracellularly. In biopsied muscle specimens, GLN was localized in CD3 positive cells adjacent to muscle fibers. GLN was not observed inside muscle fibers.Taken together, GLN may be released from T cells as well as perforin leading to muscle fiber damage, and muscle cells do not uptake GLN so that GLN-mediated apoptosis does not take place. Roles of interferon-α and dendritic cells for inflammatory muscle disorders Izumi Kawachi a , Keiko Tanaka a and Masatoyo Nishizawa a a Department of Neurology, Brain Research Institute, Niigata UniversityThe therapeutic administration of interferon (IFN)-α can provoke autoimmune disorders, such as sarcoidosis. Moreover, there is the evidence that IFN-α can lead the differentiation of the dendritic cells (DCs), that are the most potent antigen-presenting cells, playing a pivotal role of induction of the immune response. This study examines whether or not IFN-α and DCs play important roles in pathogeny of inflammatory muscle disorders such as sarcoidosis and polymyositis (PM). The sera of sarcoidosis contained high amounts of IFN-α, CXCL10/IP10, and interleukin (IL)12p40, while the sera of PM contained moderate amounts of CXCL10/IP10 and IL12p40, in comparison with other neurological disorders (OND) as measured by ELISA. The skeletal muscles in sarcoidosis contained considerable amounts of IFN-α and CXCL10/IP10, while PM and OND had little or no elevation of them as measured by real time RT-PCR. Notably, numerous DC-LAMP-positive cells, that had the specific marker for mature DCs, accumulated in epithelioid granulomas of sarcoidosis, whereas PM and OND had little or no DC-LAMP-positive cells by means of immunohistochemistry. These mature DCs produced Th1-chemokine CXCL10/IP10 in close proximity to CXCR3 positive cells. Several TLR stimulants can produce high amounts of IFN-α via the activation of plasmacytoid DCs. We point out the existence of a natural alliance between IFN-α and DCs development, instrumental for ensuring an efficient connection between innate and adaptive immunity.Poster Session 18: Paraneoplastic and antibody-mediated diseases one with anti-Hu-associated PNS; evidence of intrathecal Ig synthesis in all controls) were included in our study. Associated tumours were small cell lung cancer in 2/5, a carcinoid tumor of the lung and breast cancer. Oligoclonal bands were detected by isoelectric focusing. Ri-specific affinity blotting was used to assess Ri-specific bands in serum and CSF. In one patient with absence of oligoclonal bands of total IgG in CSF, anti-Ri-specific oligoclonal bands were detected with the same sample indicating a higher sensitivity of Ri-specific affinity blotting as compared to affinity blotting with anti-human IgG antibodies. Conclusion: Our findings provide further arguments that anti-Ri-specific antibodies are produced by B-cell clones in the central nervous system of patients with PND and support the hypothesis of an intrathecal immune reaction being responsible for anti-Ri associated paraneoplastic neurological syndromes. PP18-05 Autoantibody in neurologic paraneoplastic disorders Objective Neurologic paraneoplastic disorders (NPD) are uncommon; however, their diagnosis is very important for maximizing the likelihood of a favorable oncologic and neurologic outcome. There is increasing evidence that the pathogenesis of many NPD appears to be an immune reaction against antigen shared by the cancer and the nervous system. We examined autoantibodies against gephyrin, amphiphysin, voltage-gated K channel (VGKC), and GAD in 15 patients with NPD.Methods Seven patients had stiff-person syndrome, four had cerebellal ataxia, one had progressive encephalomyelitis with rigidity (PER), one had chorea, one had limbic encephalitis and one had sensory polyneuropathy.Protein extraction from rat brain was separated by SDS gel electrophoresis. Western blot analysis was performed using serum samples of 11 patients with NPD. Immunohistochemical analysis was also done using rat brain tissue.Results One SPS patient with mediastinal cancer was revealed anti-gephyrin antibody. One patient with PER and breast cancer was revealed autoantibody against amphiphysin. One patient with ataxia and small cell lung cancer was revealed autoantibody against amphiphysin. Two patients with stiff-person syndrome were revealed anti GAD antibody. One patient with chorea and lung cancer was revealed anti-VGKC antibody.Conclusion Autoantibody was detected in six patients out of fifteen. The identification of antibodies in the serum of NPD patient confirms the clinical diagnosis of paraneoplastic syndrome, and allows early identification of an underlying tumor. PP18-06 Absence of paraneoplastic antineuronal antibodies in sera of 148 patients with motor neuron disease Oliver Stich 1 , Barbara Kleer 1 , Sebastian Rauer 1, 2 Background: Isolated motor neuron disease (MND) as paraneoplastic syndrome is controversially discussed. Some rare cases suggest "concurrence" of cancer and MND. However, a large cohort of patients with motor neuron disease was never screened systematically for the prevalence of paraneoplastic antineuronal antibodies.Objective: To assess the frequency of well-characterised antineuronal antibodies among patients with motor neuron disease.Methods: We tested sera from a cohort of 148 patients with motor neuron diseases for the presence of paraneoplastic antineuronal antibodies by six different ELISAs employing recombinant antigens (HuD, Yo, Ri, CV2, Ma2, Amphiphysin).Results: No patients with high titre of antineuronal antibodies suggestive of paraneoplastic origin were detected. Only 5 patients revealed low concentrations of antineuronal antibodies in sera. Confirmation test with immunoblot as well as analysis of intrathecal antineuronal antibody synthesis in these cases were negative.Conclusion: We do not found a correlation between motor neuron disease and the presence of antineuronal antibodies suggestive for a paraneoplastic etiology of MND. PP18-07 Recombinant immunoblot for the detection of paraneoplastic antineuronal antibodies specific for HuD, Yo, Ri, CV2 (CRMP5), Ma2 or amphiphysin Francesc Graus 1 , Christiane Rasiah 2 , Sebastian Rauer 2, 3 Background: The aetiology of limbic encephalitis (LE) has been unclear. Anti-Voltage-gated potassium channel antibodies (VGKC-Abs) have recently been reported in some cases of reversible LE. VGKC-Absassociated LE is potentially treatable encephalitis that can be diagnosed by a serological test. We also evaluated the electrophysiological influence of anti-VGKC antibodies of LE patients. Methods: Sera were obtained from consecutive 20 LE patients, consecutive 20 acquired neuromyotonia (NMT) as positive controls, and 20 healthy controls. Serum VGKC-Abs titers were measured by radioimmunoassay using whole rabbit-brain homogenate and 125 I-aDTX. IgG were purified with HiTrap ProteinG and HiTrap Desalting column for following electrophysiological study (patch-clamp). To study the effect of the patients' IgG on the electrical properties of NB-1, we cultured the cells with 5 μg/ml IgG from the patients or controls for 3 days. In K + current recording, cells were held at the holding potential of − 80 mV, and square pulses (300 ms duration) between − 140 and +60 mV (20 mV steps) were applied at 15 s intervals. There was no difference of clinical features between anti-VGKC Abs positive and negative LE. Anti-VGKC Abs of NMT suppressed potassium cation currents on NB-1 cell; meanwhile Abs of LE did not. CONCLUSIONS: There was no difference of clinical presentation between anti-VGKC associated LE and not associated LE. Further studies are necessary. PP18-09 Potassium channel antibody specificities in different neurological syndromes Antibodies to potassium channels, specifically the Kv1.1, 1.2 and 1.6 subtypes, are being detected in a growing number of patients with neurological diseases. The patients present mostly with peripheral nerve hyperexcitability (neuromyotonia with muscle cramps and fasciculations), with central nervous system involvement (limbic encephalitis with memory loss and seizures), or with combinations of these and autonomic symptoms (Morvan's syndrome with additional sleep, cardiac and gut dysfunction). The antibodies are detected by radioimmunoprecipitation of 125 I-dendrotoxin labeled VGKCs extracted from mammalian brain tissue, and their titers range between 100 and 7000 pM. Since dendrotoxin binds to VGKCs Kv 1.1, 1.2 and 1.6, it is not clear whether the patients' antibodies bind to distinct Kv subtypes, and whether the antibody specificities correlate with the different clinical syndromes. We have characterized the antibodies by binding to different parts of the nervous system, comparing with commercial antibodies to the various Kv1 subtypes. We found that IgG antibodies in patients with limbic encephalitis co-localise with antibodies to Kv1.1 in peripheral nerve, spinal cord, cerebellum and the hippocampus, whereas antibodies in patients with neuromyotonia mainly co-localise with Kv1.2 in these tissues. These results have been confirmed on cell lines expressing the individual VGKC subtypes. The results suggest for the first time that the clinical expression of VGKC-antibody associated diseases correlates with antibodies specific for a VGKC subtype, and will inform further experimental studies. A set of cancer and systemic autoimmune diseases is accompanied by an autoantibody response to nucleolar protein B23/nucleophosmin. Probably the appearance of B23 autoantibodies is induced by a unique B23 conformation in tumor cells. Thus, it has been demonstrated that the unique B23 conformation in liver tumor likely results from an N-terminal truncation of B23 present in tumor hepatocytes that forms SDS-stable, GB-sensitive oligomers (Ulanet D.B. We established the structural state of nucleophosmin in different human tumor cells (HeLa, NGP, Hep G2, Jurkat, OSA) and normal mouse tissues (brain, lung, kidney, liver). The SDS-stable oligomeric forms of nucleophosmin were observed exclusively in the tumor lysates. Interestingly, the number of SDS-stable oligomers and their electrophoretic mobilities were different in examined cells. We have also defined the character of N-terminal truncation in HeLa cells. This study was supported by the Russian Foundation for Basic Research (project 06-04-48388) PP18-11 Serum anti-CNS antibodies are common in chronic HLA DR2 + narcolepsy/cataplexy Introduction: HLA associations and other findings suggest that sporadic narcolepsy/cataplexy (N/C) may be autoimmune-mediated, especially in DR2 + ve hypocretin-deficient patients.Objective: To look for serum CNS antibodies in DR2 + N/C. Methods: We tested 46 sera-29 DR2 + N/C (17 f, age 43 ± 16 y; duration of N/C 19 ± 18 yrange 1-55 y); 11 other sleep disorders (OSD; 5 f, age 41 ± 13); and 6 healthy (3 f, age 39 ± 4)by immunohistochemistry (IHC; 1:500) on rat brain and Western blotting (WB; 1:50). All individuals tested negative for other autoantibodies including onconeural, ANA, ds DNA and ENAexcept 2 N/C with thyroid ab + ve hypothyroidism. 4/4 N/C and 2/2 OSD patients tested within 5 y of disease-onset were VGKC ab − ve.Results: CNS ab + ve: N/C 62% (18/29 + ve − 8/29 IHC, 3/29 WB, 7/29 both); OSD 45% (5/11 + ve − 2/11 IHC, 1/11 WB, 2/11 both); healthy 16% (1/6 + ve − 1/6 IHC). All N/C serum binding was weak to moderate. 3 N/C, but none of the control, sera bound to some brainstem neurones on IHC and 50-60 kDa proteins on WB. There were no other consistent patterns within or between groups. Within the N/C group, ab positivity did not correlate with time between syndrome onset and serum testing.Conclusions: Low-titre serum CNS abs are common in chronic N/C and are probably disease non-specific. Our findings do not exclude autoantibodies from the pathogenesis, but should be considered when designing future studies. Ideally, antibody testing should be close to N/C onsetas a diseasespecific antibody response may be monophasic and short-lasting. PP18-12 Anti-gliadin antibodies in celiac disease target the C domain of synapsin I Armin Alaedini, Norman Latov Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, NY, USA Neurologic deficits, including neuropathy and ataxia, are among the most commonly associated extraintestinal complications of gluten sensitivity, although little is known about their pathogenesis. A mechanism of autoimmunity resulting from molecular mimicry between gliadin and a nervous system autoantigen has been suspected. Previously, we showed that anti-gliadin antibodies in celiac disease bind to synapsin I, a neuronal phosphoprotein involved in the regulation of neurotransmitter release. In this study, we sought to further characterize the immune cross-reactivity by determining the epitopes of synapsin I involved in the interaction with antigliadin antibodies. Antibodies were affinity-purified from pooled sera of multiple gliadin-immunized rabbits by column chromatography. The reactive epitopes of synapsin I were mapped by assessing the binding of purified antibody to arrays of overlapping human synapsin Ia/Ib peptides synthesized on cellulose membrane. The designed peptides were 14mers, with an offset of 6 amino acids each. Major immunoreactive epitopes were found to be located in the C domain of synapsin I, corresponding to sections of the protein that are surface-exposed and directed away from the plane of dimerization. These epitopes also form part of the domains shown to have a role in mediating the interaction between synapsin and synaptic vesicles by penetrating into the lipid membrane. The findings in this study suggest that synaptic uptake of anti-gliadin antibodies that bind synapsin I may have a deleterious effect on the regulation of neurotransmitter release, and might be involved in the pathogenesis of the neurologic manifestations of gluten sensitivity.manifestations of CNS (central nervous system) engagement is variable, it ranges from migraine to severe psychotic conditions. A MS (multiple sclerosis) like syndrome is seen in some cases. Since SLE (systemic lupus erythematosus) is the prototype of B cell mediated autoimmune disease and MS is regarded as being T cell mediated, it is of interest to compare the neuroinflammatory mechanisms in the two diseases. BAFF (B-cell activating factor) and APRIL (a proliferation-inducing agent) are relatively recently described proteins, members of the TNF (Tumor Necrosis Factor) super family and of importance for B cell mediated inflammation.Methods: 31 NPSLE patients, all females, participated in the study. They were neurologically examined, cognitive tests and MRT (magnetic resonance tomography) were performed, CSF (cerebrospinal fluid) and peripheral blood were collected. BAFF and APRIL protein levels in CSF and blood were analyzed using commercially available ELISA kits. For comparison, samples from MS patients and other non-inflammatory neurological diseases (OND) from a biobank were used.Results: We found marked elevated levels of APRIL in CSF among the NPSLE cases compared to MS and OND. Blood levels of BAFF were increased in NPSLE, but in the CSF no significant differences between the groups could be detected.Conclusions: We here for the first time present CSF levels of BAFF and APRIL from NPSLE patients. magnus.la.andersson@karolinska.se PP18-17 Interleukin-6 in neuro-Behcet's disease: Association with long term outcome Interleukin-6 (IL-6), a cytokine of mainly innate immunity, is secreted by mononuclear phagocytes, endothelial cells, and activated astrocytes. One of the major functions of IL-6 is the stimulation of neutrophils. It has been reported that cerebrospinal fluid (CSF) IL-6 is increased in progressive neuro-Behcet's disease (BD). Here we aimed to assess any relationship of CSF IL-6 with long term prognosis of neuro-BD. A total of 68 patients (43 males) with BD and various forms of neurological symptoms were included: 13 had primary headache disorders, 40 had parenchymal neuro-BD, 10 had dural sinus thrombosis, and 5 had stroke. Serum and CSF IL-6 levels were assessed by ELISA. There was no significant difference in serum levels of IL-6. Mean CSF IL-6 levels (pg/ ml) were, 4.9, 15.6, 84.3, and 12.1 in the groups with headache, sinus thrombosis, parenchymal neuro-BD and stroke, respectively (p < 0.01). IL-6 levels were increased if the patient was examined at an acute attack, and were correlated with CSF cell counts and total protein levels. When the patients that were independent after at leas 3 years were compared to those that were dead or dependent, increased IL-6 levels were found in the second group; however patients with an elevated CSF cell/protein level at the acute stage also had significantly higher rate of long term disability (p < 0.05). Although elevated CSF IL-6 levels seem to be related to long term disability, still CSF cell/protein levels seem to be good markers, cheap and easy to perform. PP18-18 Longitudinal study of patient with neuro-Sweet disease presenting with recurrent encephalomeningitis A. Kimura, T. Sakurai, A. Koumura, Y. Suzuki, Y. Tanaka, I. Hozumi, T. Inuzuka Department of Neurology and Geriatrics, Gifu University Graduate School of Medicine, Gifu, Japan Background: Neuro-Sweet disease (NSD) has recently been reported as Sweet disease with central nervous system (CNS) involvement characterized by multisystem neutrophilic infiltration. Skin biopsy histology shows a dense dermal infiltration of neutrophils with no signs of vasculitis, which is important in distinguishing NSD from neuro-Behçet disease in addition to HLA B51 negativity.Method: We longitudinally analyzed the clinical, laboratory and neuroimaging data of a patient with NSD for one year.Results: The patient presented with encephalomeningitis twice in one year. The histology of a skin biopsy obtained from an erythematous plaque of the left upper limb, was compatible with that of NSD. In the acute phase of encephalomeningitis, peripheral blood neutrophil and Creactive protein levels increased. Small increases in protein levels and the degree of pleocytosis with a predominance of mononuclear cells were observed in CSF. The investigation of cytokine levels in CSF showed increases in the levels of IL-6 and IFN-γ in the acute phase. On HLA typing, B54 and Cw1 were positive, but B51 was negative. Brain MRI T2WI showed high-signal-intensity lesions in the brainstem, thalamus, caudate nucleus, cerebral subcortex, and cerebral cortex in the acute phase. Systemic glucocorticoids were effective in improving the neurologic symptoms, and the abnormalities of MRI and laboratory findings. However, encephalomeningitis recurred after glucocorticoid therapy was discontinued.Conclusion: The relationships of the levels of IL-6 and IFN-γ with encephalomeningitis disease activity in NSD were found. It is important to develop a monitoring marker of disease activity and prophylactic therapies. PP18-19 Collagenous tissue disease and CNS involvement Purpose: Vasculitis of the central nervous system (CNS) is classified as primary angiitis or as vasculitis secondary to a variety of diseases. The aim of this study is to determine the characteristics of the patients who were initially presented with CNS syndromes and diagnosed as CNS involvement of collagenous tissue disease after a serial examination.Methods: 26 patients(22 female, 4 male) who were followed up between January 1996 and September 2005 in University of Hacettepe Hospital, Department of Neurology are included. Demographic features, initial complaints, the duration between initial complaints and final diagnosis, laboratory findings, modality of imaging techniques, variety of diagnosis and differential responses to treatment are evaluated.Results: The ages of the patients ranged from 22 to 63 years. The most common presenting features were convulsion, hemiparesis and headache. 13 patients were diagnosed as SLE, 5 patients as Sjogren's syndrome, 3 patients as isolated CNS vasculitis,1 as rheumatoid arthritis and 4 patients as probable CNS vasculitis. In most cases, despite years of detailed examination, the autoantibodies remained undefined for a long time, maximum of 12 years.Cranial and spinal magnetic resonance imaging (MRI) findings have different characteristics from other demyelinating diseases. Intensive immunosuppressive therapy seems to be an effective treatment modality.Conclusion: Insistent questioning of systemic involvement and MRI examination are important for diagnosis. If a patient has a preliminary diagnosis of demyelinating disease and gives no response to immunomodulatory therapy, then one should think of CNS vasculitis. PP18-20 HLA-B54/Cw1-positive benign recurrent encephalomeningitis and possible neuro-Sweet disease K. Hisanaga a , Y. Iwasaki a , Y. Itoyama b a Miyagi National Hospital, Miyagi, Japan; b Tohoku University, Sendai, Japan Sweet disease is a multisystem inflammatory disorder characterized by painful raised erythematous plaques and aseptic neutrophilic infiltration of various organs. Skin biopsies reveal deep dermal infiltration of mature neutrophils and absence of vasculitis. Sweet disease responds to systemic corticosteroids. We reported cases of Sweet disease with neurologic manifestations as neuro-Sweet disease (NSD), and proposed a criteria for the diagnosis. Cases with benign, but frequently recurrent encephalitis or meningitis accompanied by autopsy verified-Sweet's erythematous plaques without Behcet's mucocutaneous lesions are classified as "Probable NSD" in the criteria. There is a strong human leukocyte antigen-B54/Cw1 association. Systemic corticosteroids are highly effective for most of the neurologic manifestations. "Possible NSD" in the criteria included HLA-B54/ Cw1-positive encephalitis or meningitis without erythematous plaques. Analysis of 7 such cases revealed that the encephalitis or meningitis were also generally benign especially compared with a relative disorder, neuro-Behcet disease with HLA-B51. Abnormalities on MRI markedly improved in most of the cases after treatment with systemic corticosteroids. It may be more acceptable to tentatively diagnose these cases as "HLA-B54/Cw1positive benign recurrent encephalomeningitis", if it is inadequate to diagnose cases without Sweet's erythematous plaques as NSD. Background: Anti-tumor necrosis factor α (anti-TNFα) therapy, including infliximab treatment, for rheumatic diseases has been associated with rare cases of new or exacerbated neuroinflammatory disorders such as multiple sclerosis and optic neuritis. In multiple sclerosis, anti-TNFα therapy may increase disease activity. There is evidence for an increased peripheral T cell reactivity, measured as enhanced IFNγ production, during anti-TNFα therapy of rheumatoid arthritis. This may potentially provoke neuroinflammation, since systemic IFNγ administration has been demonstrated to exacerbate multiple sclerosis. We therefore hypothesize that the few cases of clinical neuroinflammatory disorders observed after anti-TNFα therapy of rheumatic diseases represents the extreme end of a commonly occurring minor intrathecal immune activation, which in most cases does not give any overt neurological dysfunction. Objective and methods: To test this hypothesis, we determined the expression of IFNγ, TNFα and IL-10 mRNA in cerebrospinal fluid cells and peripheral blood mononuclear cells, the levels of nitric oxide oxidation products in cerebrospinal fluid and serum, the cerebrospinal fluid cell counts and IgG indices, in ten patients with polyarthritis before and during infliximab treatment. Results: No significant signs of intrathecal immune activation were recorded. In the systemic compartment, induction of IFNγ expression during infliximab treatment was demonstrated. Conclusions: Intrathecal immune activation, as measured by the employed assays, during infliximab therapy is not a common phenomenon and we thereby refute our initial hypothesis. Our finding of increased systemic IFNγ expression demonstrates that a systemic pro-inflammatory component is part of the diverse effects of anti-TNFα therapy.The neuroinflammatory reaction has been linked with PD. Focusing on this reaction, one of the hypotheses to explain the significance of age and gender (male dominance) effects on neurodegenerative processes in PD may result from a link between these risk factors and the inflammatory processes. The aim of our study was simultaneous determinations of striatal: tyrosine hydroxylase (TH) protein concentrations and cytokines (TNFa, IFNg, IL-1b, IL-6 and TGFb 1 ) gene expression in young and aged (3 and 12 months old) C57BL/6 male and female mice after 6 h; 1, 3, 7, 14, 21 days post 1-methyl-4-phenyl-1,2,3,6 tetrahydropiridine (MPTP) intoxication. Western blotting analysis shown that in males and females the decrease in the level of TH was observed within 1-21 days following intoxication, achieving minimal level between 3 and 7 time-points. However, at the early time-points, males showed greater reduction in striatal TH versus females. RT-PCR analysis revealed that the increases in expression of TNFa, IL-1b and IFNg genes post intoxication were faster in both young and aging males than females. In aged and young males we observed maximal increase in TGFb 1 after 1-day post intoxication. In contrast, in young and aged females TGFb 1 was elevated at later time-points. MPTP caused an increase of IL-6 in striatum of males and females, but this increase was significantly higher in females.These observations may help in the better understanding the age and gender difference which exist in PD. PP19-02 Regulatory T cells show higher frequency in aged individuals and increased suppressive activity in neurodegenerative diseases For neurodegenerative disorders like Alzheimer's (AD) and Parkinson disease (PD) neuroinflammation is an established fact but the role of inflammation remains obscure. However, due to promising immunization studies in animal models for both diseases, the idea has evolved that a beneficial autoimmune response may prevent disease development. Even though this idea remains to be proven, we decided to analyze Regulatory T cells (Treg) from AD and PD patients.Our working hypothesis is that higher activity or numbers of Treg may suppress a beneficial immune response, resulting in, or accelerating, either AD or PD. Four different groups have been analyzed: AD and PD patients as well as healthy young and aged-matched non-demented individuals.Our findings indicate that all elderly groups have a higher Treg (CD4 + Foxp3 + ) frequency compared to the young group. Additionally, we observed a trend of increasing Treg activity for the healthy elderly group compared to the young group, which developed into significantly higher Treg activity for AD and PD patients compared to the elderly healthy donors.The observation, of Treg differences already in the total pool of peripheral Treg between young and elderly groups, identify Treg as immunecells undergoing immunological senescence. Taking into account that changes in Treg function increase with a neurodegenerative phenotype present in AD and PD patients, our data support the hypothesis of an existing beneficial autoimmune effector mechanism in these patients, which is suppressed by Treg. Consequently, it will be of special interest to analyze Treg specific for CNS-relevant antigens in AD and PD. Parkinson's disease (PD) is a progressive neurodegenerative disorder of unknown etiology, which is characterized by loss of nigrostriatal dopamine (DA) neurons, accompanied by DA deficiency in the striatum. The degenerative processes observed in the nigrostriatal system lead to neuroinflammatory response results in glial activation manifested by elevated inflammatory mediators including cytokine and nitric oxide (NO).In the present study we examined iNOS (inducible nitric oxide synthase) protein expression and neurotransmitters levels in the striatum of C57BL/6 male and female mice (2 and 12 months old) in a murine model of PD induced by MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). iNOS protein expression was assayed by Western Blot method. The relatively quick rise of iNOS expression in group of young and old male mice at 6 h-21 days was observed. While in young female mice at 1-7 days old and on the 3rd to the 21st days such increase of iNOS expression was detected.Additionally using HPLC method we examined levels of selected neurotransmitters. The significant decreased DA level was observed in female within 1-21 days and in male within 6 h-21 days following MPTP injection.Correlation analysis revealed that in whole study groups a negative correlation between iNOS protein expression and DA concentration as well as a positive correlation between iNOS protein expression and DA turnover (HVA/DA) were found.Factor analysis allowed for extraction of independent factor, representing of biochemical data, indicating on central neuroinflammatory process engaged in increased iNOS expression and simultaneous decreased of DA concentration. Aim of the study was to evaluate the difference in cytokine production by mononuclear cells (PBMC) from healthy controls (HC), AD and PD patients. Methods: The subjects were composed of 3 groups as follows: 16 AD patients with clinical diagnosis of according to DMS IV-R and NINCDS-ADRDA; 16 patients with idiopathic PD according to the London Brain Bank Criteria and 16 HC, matched for sex and age. None of the patients had a history of infectious, immune, or any other neurodegenerative disorders. PBMC were purified and cultured without or with PHA (20 μg/ml). MCP-1, RANTES and IL-1 levels in 24-h supernatants were measured with commercially available ELISA kits. Statistical analysis was performed using Wilcoxon test. Results and discussion: Our present study suggest that MCP-1, RANTES and IL-1b production by unstimulated and PHA stimulated PBMC from PD or AD where significantly different from those by PBMC from HC (p < 0.01). This could provide new insight into immunoregulatory characteristics two different neurodegenerative disease. The use of PBMC as a peripheral model for in vitro/ex vivo may help studies on the pathogenetic mechanism and on treatment effects in PD and AD. PP19-09 Amyloid β protein 1-38 (Aβ38), Aβ40 and Aβ42 levels in matched CSF and Alzheimer disease (AD) Objective: To quantitate the levels of Aβ38, Aβ40 and Aβ42 in matched pairs of cerebrospinal fluid (CSF) and plasma from patients with Alzheimer disease (AD) and non-AD controls, and correlate the data with age, and ApoE genotype.Background: Investigators reported that Aβ38 levels were higher than Aβ42 in CSF, and it was the second most prominent Aβ species next to Aβ40. However, levels of Aβ38, Aβ40 and Aβ42 were not measured in matched CSF and AD plasma.Methods: The study included 32 patients (20 women and 12 men) with probable AD (mean age 71 ± 1 years) and 25 (16 women and 9 men) elderly nondemented controls (72 ± .3 years). Levels of Aβ38, Aβ40 and Aβ42 were measured using ELISA.Results: CSF Aβ38 and 40 levels were similar between AD and controls, but Aβ42 were significantly lower in AD than controls (p = .001). Plasma levels of Aβ38, 40 and 42 in both groups were similar. CSF Aβ38 and 40 levels were higher in controls with ApoE4 than those without. There was a positive correlation between age and plasma Aβ40 (p = .01) in AD group. There was a strong correlation between CSF Aβ38 and Aβ40 levels (p = .001). There was no correlation between levels of CSF and plasma Aβ38, Aβ40 or Aβ42 in both groups.Conclusion: A combination of Aβ species measurements in CSF is useful to aid the diagnosis of AD. A lack of relation between CSF and plasma Aβ species indicate that the source of synthesis of Aβ is different. PP19-10 Inflammation in CNS increases Alzheimer's disease related neurofibrillary-tangle burden: A study of experimental autoimmune encephalomyelitis in a transgenic animal model for neurofibrillary-tangles Occurrence of T cells in the substantia nigra of patients with dementia with Lewy bodies Neuroinflammation plays a significant role in some neurodegenerative diseases. Involvement of the brain-derived, innate immune system components such as microglia and complement proteins seems to be established in the last two decades. On the other hand, significance of the acquired immune system, where T cells play a central role, has yet to be unveiled or, even, hardly investigated. We here report the occurrence of T cells in the substantia nigra (SN) of patients with dementia with Lewy bodies (DLB). In the SN of many DLB patients, a number of melanin positive cells still remain and active neurodegenerative processes are seen even at the time of death. Immunohistochemistry for CD3 has revealed that more T cells than controls are present in the blood vessels and brain parenchyma in the SN of many, but not all, DLB patients. The number of T cells was not related with the degree of systemic inflammation at the agonal stage or with the number of T cells in the hippocampus of the same patient. Relationship to the degree of alpha-synuclein accumulation was not evident. Our results suggest that a small number of T cells enter the substantia nigra if the process of neurodegeneration is vigorous and microglia is highly activated. Whether such T cells contribute to the pathophysiology of nigral degeneration is an issue of future investigation.Poster Session 20: Ischemia, injury and brain tumor Endothelin-1-induced focal cerebral ischaemia: Neuronal death in the absence of an inflammatory response L. M. Felton a , S. Waters a , V. H. Perry a a CNS Inflammation Group, School of Biological Sciences, University of Southampton, Southampton, UKThe aim of this study was to characterise the inflammatory response to a novel in vivo model of focal cerebral ischaemia induced by an intrastriatal injection of the potent vasoconstrictor substance endothelin-1 (ET-1) 1 .Focal cerebral ischaemia was induced in rats by an intrastriatal injection of 10 pmol ET-1; injections were also made in combination with recombinant interleukin-1β (IL-1β; 1-50 ng). Neuronal death, leucocyte recruitment and cytokine expression was measured using immunocytochemistry, Taqman PCR (mRNA) and ELISA (protein).Injection of ET-1 gave rise to a localised neuronal loss (lesion volume 3.1 ± 0.8 mm 3 ) and minimal leucocyte recruitment at 24 h. Expression of IL-1β, interleukin-6, tumour necrosis factor-α, cyclooxygenase-2 and transforming growth factor-β1 was either low or undetectable; only expression of the anti-inflammatory mediator interleukin-1 receptor antagonist was increased at mRNA and protein levels. Co-injection of IL-1β with ET-1 failed to modify lesion volume for up to three days, despite inducing a dose-dependent increase in neutrophil recruitment.We confirm that focal cerebral ischaemia induced by an injection of ET-1 gives rise to a minimal inflammatory response, and demonstrate that in contrast to previous studies showing that IL-1β exacerbates ischaemic and excitotoxic brain injury, this cytokine has no impact on ET-1-induced cerebral ischaemia.Funded by BBSRC and Nurin Ltd. 1 Hughes et al. PP20-02 Interleukin-1β is expressed by microglia and blood-borne macrophages after focal cerebral ischemia in mice Abstract -Interleukin-1beta (IL-1β) is known to play a central role in ischemia-induced brain pathology. Using a murine model of focal cerebral ischemia we have recently shown accumulation of large numbers of IL-1β mRNA-expressing and IL-1β immunoreactive cells in the periphery of the infarct. This might reflect induction of IL-1β in resident microglia, recruitment of blood-borne IL-1β producing cells, presumably blood-borne macrophages or a combination thereof (Clausen et al., 2005) . To address this question, we generated green flourescens protein (GFP + ) bone marrow chimeric mice. Focal cerebral ischemia was induced by permanent occlusion of the middle cerebral artery (MCA). Cell counting revealed thousands of donoer-derived GFP + cells within the infarct and peri-infarct 24 h after MCA occlusion, most of these cells displayed a round or elongated morphology and expressed the microglial-macrophage antigen Mac-1. At this time of observation, no bone marrow-derived cells had transformed into ramified microglial-like cells. Triple-immunofluorescent analysis showed that IL-1β protein was located only to infiltrating a small proportion of infiltrating blood-borne macrophages (Mac-1 + GFP + Mac-1 + GFP + ) within the infarct and peri-infarct and to activated resident microglia (Mac-1 + GFP − ) in the peri-infarct expressed IL-1β protein after 24 h of permanent MCA occlusion. At the same time, very few activated microglia (Mac-1 + GFP − ) located in the periphery of the infarct synthesised IL-1β + protein.In summary, the data show that permanent occlusion of the MCA in mice results in expression of IL-1β protein in segregated subpopulations of both microglia and macrophages. PP20-03 The role of the surfactant protein D in ischemic brain injury and inflammation in mice Surfactant protein D (SP-D) is a member of the collectin family of collagen-like lectins. SP-D is an endogenous modulator of inflammation and binds to pathogenic microorganisms and apoptotic cells. SP-D is present in the circulation, synthesized by endothelial cells and macrophages, and has pro-atherogenic effects in mice. To investigate whether SP-D also plays a role in stroke-induced infarction or in the secondary ischemia-induced inflammatory responses, we subjected SP-D knockout and wildtype (WT) mice to permanent occlusion of the middle cerebral artery (pMCAO). In this model, the acute infarction, results in breakdown of the blood-brain barrier and a predictable microglial-macrophage-reaction. Comparison of mean infarct volumes after 24 h survival revealed no distinction between the knockout and the WT mice. Quantitative PCR showed an ischemia-induced upregulation of the mRNA levels of IL-1beta and TNF which, however, was similar in knockout and WT mice. Immunohistochemical analysis for microglial-macrophage Mac-1 showed activation of microglia and macrophage infiltration into infarct and peri-infarct in both groups of mice. The data suggest that SP-D has no effect on ischemia-induced brain injury and neuroinflammation at 24 h after surgery. Studies in progress on mice with 5 days survival will tell if systemic or locally produced SP-D affects the inflammatory responses in the protracted phase following experimental stroke. PP20-04 Ischemic neuron-derived conditioned media protect ischemic brain in ERK and NO related manner Cerebral ischemia-induced brain inflammation often leads to brain cell death and function deficit of stroke patients. The main purpose of the study was to evaluate the anti-inflammatory action of conditioned media (CM) of ischemic neurons in cerebral ischemia. In the study, protective effect of neuron-derived CM was determined both in vivo and in vitro. The CM was collected from a neuronal culture deprived with glucose, oxygen and sera (in vitro ischemia) for 6 h. At the end of 90 min bilateral common carotid artery occlusion plus unilateral middle cerebral artery occlusion (CCAO/MCAO) CM was injected directly into the ischemic brain of S.D. rat followed by 24 h reperfusion. Brain infarct size was then determined. CM-mediated protection of ischemic microglia, astrocytes and neurons were also determined in vitro based on the survival rate, phosphorylation of extracellular signal regulated kinase (ERK), expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) release of these cells with or without CM. Results showed that CM significantly reduced the infarct size of ischemic brain and increased survival of ischemic brain cells of all three types. Ischemia-induced iNOS expression and NO release were suppressed by CM in ischemic microglia and astrocytes but not neurons. On the contrary, ERK phosphorylation was further increased by CM in all three cell types. These results indicate ischemia-induced brain inflammation and cell death could be prevented by ischemic neuron-derived CM that further suggests a therapeutic value of this CM in future control of cerebral ischemia. PP20-05 ADAMTS-1 and -4 are up-regulated following transient middle cerebral artery occlusion in the rat and their expression is modulated by cytokines in astrocytes and endothelial cell cultures Cross AK 1,2⁎ , Haddock G 1,2 , Stock CJ 3 , Allan S 3 , Surr J 1,2 , Bunning RAD 1 , Buttle DJ 2 , Woodroofe MN 1 1 Biomedical Research Centre, Sheffield Hallam University, Howard St, Sheffield, S1 1WB, UK; 2 Division of Genomic Medicine, E Floor Medical School, University of Sheffield, Sheffield S10 2RX, UK; 3 Faculty of Life Sciences, The University of Manchester, Michael Smith Building, Manchester, M13 9PT, UK ADAMTS (a disintegrin and metalloproteinase with thrombospondin motif)-1, -4 and -5 enzymes are secreted metalloproteinases which bind to the extracellular matrix (ECM) and substrates include the brain ECM components: aggrecan, versican and brevican. Their main inhibitor is TIMP-3.Focal cerebral ischaemia leads to neuronal damage and infiltration of the ischaemic area by inflammatory cells. In the present study focal cerebral ischaemia was produced by temporary occlusion of the middle cerebral artery (MCAO) in the rat. Brain tissue was obtained at 6 h, 24 h and 5 days after MCAO and ADAMTS-1, -4 and -5 and TIMP-3 mRNA and protein expression was measured.ADAMTS-1 and -4 mRNA levels were increased in the occluded hemisphere compared to the contralateral hemisphere of MCAO animals and brain tissue from sham-operated rats, 24 h post MCAO. This was accompanied by an increase in mRNA levels for interleukin (IL)-1β, IL-1 receptor antagonist (IL-1ra) and tumour necrosis factor (TNF). ADAMTS expression was modulated by TNF and IL-1 in primary human astrocyte and brain endothelial cell cultures. Increased ADAMTS-1 and -4 in MCAO may be detrimental, promoting ECM breakdown and enabling infiltration of inflammatory cells. In contrast, beneficial effects may include the promotion of neurite outgrowth by removal of inhibitory ECM molecules. PP20-06 Activation of vessels and glial cells in the rat brain under chronic cerebral hypoperfusion a Hideaki Wakita, a Kaichi Yoshizaki, c Hidekazu Tomimoto, a Keikichi Takahashi and b Takeshi Tabira a Department of Vascular Dementia Research, b National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Obu City, Japan; c Department of Neurology, Graduate School of Medicine, Kyoto University, Kyoto, JapanRarefaction of white matter and impairment of memory function have been reported in the rat under chronic cerebral hypoperfusion. Using this model, we investigated the activation of vessels and glial cells in the white matter lesions. Male Wistar rats were subjected to the permanent occlusion of the bilateral common carotid arteries. Glial activation was examined with immunohistochemistry for major histocompatibility complex (MHC) class II antigen and glial fibrillary acidic protein (GFAP).Activation of vessels and glial cells were then assessed up to 30 days after the occlusion. In sham-operated animals, TNF-α and CD62E were expressed in only a few vessels. GFAP immunopositive astrocytes were distributed predominantly in the white matter. After occlusion, the number of vessels immunopositive for TNF-α and CD62E increased in the white matter including the optic tract, corpus callosum and fiber bundles of the caudoputamen. The number of MHC class II antigen-positive microglia and GFAP-positive astrocytes increased preferentially in the white matter. These changes persisted even after 30 days of occlusion. Rarefaction of white matter was observed in the same regions. These results indicate that persistent activation of vessels and glial cells may play a role in the pathogenesis of white matter rarefaction under chronic ischemia. This suggests an involvement of immunological and inflammatory pathomechanism in the cerebrovascular white matter lesions. PP20-07 Differential regulation of prostaglandin E 2 receptor subtypes in the rat hippocampus after cerebral ischemia and ischemic tolerance We investigated the distribution and time course of expression of two subtypes of prostaglandin E 2 (PGE 2 ) receptors, EP2 and EP4, in a rat model of cerebral ischemia and ischemic tolerance. A short (3 min) cerebral ischemia as well as a three-minute ischemia followed by a second lethal ischemia enhanced the expression of EP2 and EP4 receptors in CA1 pyramidal neurons of the hippocampus. In tolerance-acquired CA1 neurons, the immunoreactivities of EP2 and EP4 were upregulated after 4 h and 12 h, respectively. The immunoreactivities were most prominent at three days, and were sustained for at least 14 days, consistent with the results of immunoblotting experiments. In contrast, immunoreactivities for these PGE 2 receptors increased in reactive glial cells in the vulnerable CA1 and hilar regions of rats subjected to the lethal ischemia without ischemic preconditioning. Most of the EP2 immunoreactivity occurred in microglial cells and some astrocytes, whereas increased immunoreactivity for EP4 occurred only in astrocytes. These data suggest that ischemia and induction of ischemia-tolerance have different regulatory effects on the expression of EP2 and EP4 receptors, and that PGE 2 may exert its unique pathophysiological functions in relation to delayed neuronal death and ischemic tolerance induction in the rat hippocampus via specific PGE 2 receptors. This research was supported by a grant (M103KV010019 04K2201 01930) from Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology of Republic of Korea. PP20-08 Spatiotemporal expression of osteopontin in rat astroyctes and microglial cells following transient forebrain ischemia Osteopontin (OPN) is an adhesive glycoprotein containing the peptide sequence Arg-Gly-Asp (RGD), which is intimately associated with cellmediated immunity. Moreover, OPN induction has been detected in a set of macrophages after focal and global forebrain ischemia and it suggests that OPN may play a key cytokine in tissue repair and remodeling within the central nervous system. We have investigated here, using in the semi-quantitative RT-PCR analysis, in situ hybridization histochemistry and immunohistochemistry, the spatiotemporal expression of OPN in the rat hippocampus following transient forebrain ischemia. Ischemia-induced OPN mRNA and protein expression appeared in a subset of microglial cells as early as 1 d, and then reached a peak at 3 d postischemia, and returned to basal levels at 7 d. However, at 10 days, OPN expression in the hippocampal CA1 region re-increased in some reactive astroglial cells, and was sustained for 14 d. Expression of OPN in the reactive astrocyte from 10 d post-ischemia is significant results as compared with established papers. These data suggest that OPN may play a key role as a chemotactic factor for astrocytes and microglial cells. In vivo imaging of phagocytic cells is gaining interest for the study of various pathologies in which they are critically involved, such as neurodegenerative diseases, brain tumors, and stroke. We present here the first MRI (Magnetic Resonance Imaging) data obtained in mice with focal cerebral ischemia, showing in vivo labeling of phagocytes with ultrasmall superparamagnetic particles of iron oxide (USPIO). USPIO-enhanced MRI kinetic analysis unravelled an inflammatory response surrounding the ischemic lesion as well as in the contralateral hemisphere through the corpus callosum. These imaging data were correlated with histochemical analysis showing inflammation remote from the lesion and ingestion of nanoparticules by microglia/macrophages. This study shows that MR-tracking of phagocytic cells is feasible in mice, which may have central therapeutic implications in light of the potential neurotoxicity of activated microglia/ macrophages during central nervous system (CNS) disorders. Toll-like receptors (TLR) have an essential role in response to microbial agents and induction of inflammatory responses. Recent reports have indicated that TLRs, such as TLR2 and TLR4 could also regulate inflammatory and immune responses by endogenous stimuli. In this study, we suggest that TLR2-dependent inflammation can be induced by the endogenous stimulation caused by the physical injury in a mouse brain. We used the stab wound method using a stereotaxic injector with a 26-G of disposable syringe to injure the brain physically. The glial cells of the wild type mouse brain were activated by the stab wound as the results of immunostaining for GFAP and CD11-b. However, in the TLR2 knock out mouse, the activation of glia was down regulated than in wild type. The same result was shown on the mRNA expression for GFAP and CD11-b after stab wound in the brain. These results briefly indicate TLR2 has a role in mediating inflammatory responses to endogenous factors in a mouse brain. To investigate the influence of ONTDs on the patterns of glial differentiation, ONTDs were induced by surgery using Hamburger and Hamilton stage 18 or 19 chick embryos. The spinal cord tissues on postoperative days (POD) 5, 7, 10, and 14 were processed to observe astrocytic, radial glial, and microglial differentiations by glial fibrillary acid protein (GFAP), vimentin and ricinus communis agglutinin-I (RCA-I) stainings, respectively. Four embryos were assigned to subgroups of each POD and control embryos. In the control group, GFAP positivity was shown faintly at the dorsal midline on embryonic day (E) 10 (corresponding to POD 7), in the ventral one-third of the white matter on E 13 and in the whole white matter on E 17. Embryos with ONTDs showed earlier and stronger GFAP positivity from POD 7-14, especially at the dorsal surface and the adjacent gray matter. In the control group, vimentin staining demonstrated a positive reaction at the midline with positivity in a faint, radial pattern on E 8 and E 10. In embryos with ONTDs, vimentin positivity was enhanced and persisted from POD 5-14. These findings were prominent along the dorsal surface of ONTDs. No difference in RCA-I staining was found between the control and ONTD groups. The results reveal that ONTD promotes astrocytic differentiation and prolongs expression of radial glial fibers, which seems to be a reaction to the damage caused by exposure of the spinal cord tissue to amniotic fluid. PP20-12 Cross-presentation of exogenous antigens by mouse microglia S. Donnou, C. Beauvillain, U. Jarry, P. Jeannin and D. Couez INSERM U564, Angers, France Microglial cells are major immunocompetent cells and function as sentinels of the central nervous system. Following any injury of the nervous parenchyma, they rapidly activate and have the ability to become competent antigen presenting cells. Several groups also demonstrated their ability to differentiate into dendritic cells. One of the most striking property of dendritic cells is their ability to cross-present antigens to CD8 + T lymphocytes. We thus asked if mouse microglial cells were able to crosspresent antigens. We compared the ability of a microglial cell line, primary adult quiescent microglial cells and neonatal microglia. All three cell types are able to activate a CD8 + antigen specific T cell line, but with variable efficiencies as primary microglial cells are indeed less efficient than neonatal cells. Interestingly, it reflects their respective activation state, confirming that adult quiescent microglia needs some activating stimulus to become fully competent. Nevertheless, we also confirmed that these quiescent microglial cells are able to take up, process and present antigens in MHC class I molecules in situ. These results thus open new perspectives in the treatment of cerebral malignancies as new glioma expressed antigens are regularly described. PP20-13 Immunotherapy of tumors with intratumoral administration of capsaicin Jaqueline Beltran, Amiya Ghosh, Pramod Srivastava, Sreyashi BasuCenter for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, MC1601, Farmington, CT 06030-1601, USA Red chili peppers (Capsicum frutescens) are highly consumed spices throughout the world. Its principal pungent ingredient is the phenol capsaicin (8-methyl-N-vanillyl-6-nonenamide) (CP). We have observed that dendritic cells (DCs), a key cell type in immune responses, have the receptor for CP, and engagement of this receptor has powerful immune consequences. In this study we demonstrate that intratumoral administration of CP into preexisting tumors results in retarded progression of the injected tumor and leads to significant inhibition of growth of other, un-injected tumors in the same animal. Further this immunity is T cell mediated and tumor-specific. These results reflect the immunological potency of a neurological ligand in modulating immune response against a pre-existing tumor. PP20-14 Role of PARP-1 in the genesis and development of the oncogenic process The cellular responses to DNA damage play an important role in tumorogenesis. Poly(ADP-ribose) synthesis is one such cellular DNA damage responses. Poly(ADP-ribose) polymerase (PARP-1) acts as a DNA damage sensor and facilitates the access of other DNA repair factors to the site. With this purpose we characterized how PARP-1 deficiency would affect the cellular response to a specific oncogenic event. PARP-1 null astrocytes showed a lower rate of proliferation than wild-type cells, maintaining this difference even in the presence of the oncogenic insult. Unexpectedly, glial cells differed from other cell types and neither of the two populations showed features of cellular senescence after an oncogenic stress. Expression of PARP-1 in Adprt −/− astrocytes rescued the wild-type phenotype. These results allow us to conclude that PARP-1 absence seems to influence, in a negative way, the cell cycle progression, especially in the presence of an oncogenic event. Thus, deficiency in PARP-1 is a proliferative disadvantage for certain tumor cells. To investigate the involvement of galectin-3, a novel substrate for matrix metalloproteinases (MMP)-2 and -9, in the process of neurodegeneration in prion disease, the expression and cellular localization of galectin-3 in association with MMP-2 and MMP-9 were studied in the brains of a mouse model of prion disease (scrapie) and human Creutzfeldt-Jakob Disease (CJD). Both messenger RNA and protein of galectin-3 were significantly increased in scrapie-affected brains, particularly at the time when the abnormal prion protein, PrP Sc , began to accumulate in the brains. Immunohistochemically, galectin-3 was mainly immunostained in B4 isolectin-positive cells (presumably activated microglia), but not in astrocytes, in areas of PrP Sc accumulation and neuronal death in scrapie infected brains as well as in human brain with CJD.These findings suggest that galectin-3 in accordance with MMP-2 is activated in brain phagocytes in human CJD and in its mouse model, implying that increased expression of galectin-3 plays an important role in the neurodegenerative processes in human CJD as well as in prion disease model. PP21-02 Deficiency in Src kinases fgr, hck, and lyn impairs leukocyte recruitment and bacterial killing by preventing production of reactive oxygen species (ROS) in experimental pneumococcal meningitis The Src family kinases (SFKs) has been implicated in the regulation of integrin signalling and phagocytosis. Bacterial meningitis is characterized by the invasion of neutrophils, and production of cytokines (e.g., IL-1β, and IL-6) which cause an inflammatory response that causes to intracranial complications, such as brain edema formation and increase in intracranial pressure (ICP). Intracisternal injection of live pneumococci in wild-type mice caused a significant increase in cerebrospinal-fluid (CSF) leukocyte count and ICP 24 h after infection. In infected fgr −/− hck −/− lyn −/− mice CSF pleocytosis was significantly attenuated whereas the increase in ICP was more pronounced. Cerebral expression of IL-6, CXCL-1, CXCL-2 and G-CSF was increased in infected triple KO mice compared with infected wild-type mice. Lack of SFKs was associated with a worse clinical outcome and higher mortality rate. Moreover, bacterial titers in the brain and in the blood of infected fgr −/− hck −/− lyn −/− mice were 10× higher compared with infected wild-type mice indicating an impaired host defense. In vitro studies revealed that in fgr −/− hck −/− lyn −/− leukocytes challenged with pneumococci ROS production was completely inhibited whereas bacterial uptake (phagocytosis) was not affected. These results indicate the SFKs fgr, hck, and lyn play a pivotal role in leukocyte recruitment and bacterial clearance by activating ROS production during bacterial meningitis. PP21-03 Neurocysticercosis: Intrathecal synthesis of IgG Takayanagui OM, Minelli C, Odashima NS Department of Neurology, Faculty of Medicine at Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil Neurocysticercosis (NCC) is a serious public health problem, especially in developing countries. Sixteen patients with the active form of NCC were selected and compared with 21 patients of the control group, which included multiple sclerosis, Guillain-Barré syndrome, aseptic meningitis, ischemic cerebrovascular disorder, and chronic headache without pleocytosis. Intrathecal synthesis of IgG was studied by quantitative methods (IgG index, Tourtellotte's formula, and Reiber's diagram) and by a qualitative method (oligoclonal IgG bands) in the serum and cerebrospinal fluid. In the control group, excluding patients with multiple sclerosis and Guillain-Barré syndrome, intrathecal synthesis of IgG was detected in 2 (11.8%) patients. The intrathecal synthesis of IgG in NCC patients had the same pattern of multiple sclerosis patients. Further studies are required to investigate possible intrathecal synthesis of IgG in the different evolutionary stages of NCC. PP21-04 Generation of a mutant of Theiler's murine encephalomyelitis virus (TMEV) causing demyelination in the brain of susceptible mice T. Okuwa a , M. Takano-Maruyama a , K. Asakura a , H. Murayama b and Y. Ohara a a Department of Microbiology, Kanazawa Medical University, Ishikawa, Japan; b Division of Pathology, Sendai City Hospital, Sendai, Japan DA strain of TMEV causes demyelination with virus persistence in the spinal cords of mice during the chronic phase of infection and is thought to be an excellent animal model for multiple sclerosis (MS). However, in MS, the lesion extensively develops not only in the spinal cord but also in the brain. In this study, using site-directed mutagenesis, we try to generate a DA mutant virus developing demyelination in the brain.Leader protein (L) is non-structural and is located at the most Nterminus of the polyprotein. Using site-directed mutagenesis, we generated a mutant virus, in which serine at the amino acid position 57 within L is substituted for proline (DAL Pro ). The findings induced by DAL Pro were virologically and pathologically compared with those induced by wild type DA.The DAL Pro titer in the brain was 200-fold higher than DA at 10 and 90 days postinoculation (p.i.). Although infected cells and inflammatory cells were prominently detected in DAL Pro -inoculated mice at 10 and 90 days p.i., those were not observed in DA-inoculated mice. In addition, demyelination was observed in corpus callosum at 90 days p.i. Inflammatory reaction and demyelination were observed in the spinal cords of both DAL Pro -and DA-inoculated mice, but more severe in DAL Pro -inoculated mice.virus possessing this 0.3 kb fragment of A8 and the A8-env in the 57 background induced a high rate of spongiform neurodegeneration within 7 weeks. Studies using cultured glial cells suggest that the 0.3 kb fragment influences the expression of Env protein. Furthermore, these neuropathogenic chimerae, despite low viral replication in the brain, exhibited a stronger expression of Env protein compared with that of non-neuropathogenic viruses. PP21-09 Role of antibody in SFV-induced remyelination F. Mokhtrianr a a Immunology Research, Departments of Medicine and Microbiology and Immunology, SUNY Downstate and Maimonides medical Centers, USA SFV, an enveloped RNA virus, replicates in the brain and induces autoimmune-mediated demyelination one week after the clearance of virus, followed by complete remyelination by 35 days, and is one of the important animal models for MS. We have previously shown that demyelination, during SFV-infection, may be dependent on antibody response to myelin peptides, which mimic viral epitopes. A recent study has suggested a strong role for human peripheral blood T-cell receptor (TCR) γδ + T cells in humoral immunity and to provide B cell help for antibody production.The role of TCR γδ + T cells on SFV-induced myelin injury and repair was investigated in wild-type (WT) and TCR γδ knockout (KO) mice. Clinically, the KO mice showed more severe illness than the WT mice. Flow cytometric analysis of mononuclear cells of the brain showed that γδ + T-cells reached a peak on d7 and decreased by d21 pi, only in WT mice. TCR γδ-knockout (KO) mice, made significantly lower antibody response to E2 137-151 than WT B6 mice did, and did not show remyelination after viral clearance, as the WT mice did. These studies have indicated that γδ + T cells are the helper cells in the production of anti E2-137-151 (membrane-associated epitope of SFV) antibody. PP21-10 Astrocyte immunoreactivity in murine encephalitozoonosis E. F. Bondan a and M. A. Lallo a a University Paulista, São Paulo, BrazilEncephalitozoonosis is an increasingly important opportunistic protozoan infection in immunocompromised individuals. This study aimed to examine the astrocytic response by using Glial fibrillary acidic protein (GFAP) and Vimentin (VIM) immunostaining in an experimental Encephalitozoon cuniculi infection in the central nervous system (CNS) of immunosuppressed mice. It is known that although VIM expression progressively disappears in astrocytes after birth, reactive astrocytes may soon recover their capacity to express this protein following injury. Adult Balb-C mice were divided into 3 groups: I-mice immunosuppressed with cyclophosphamide (50 mg/kg, twice a week, intraperitoneal route-IP) during the experimental period and inoculated with E. cuniculi (IP); IIimmunocompetent mice inoculated with E. cuniculi; and III-non-infected and non-immunosuppressed mice. The infected animals were killed from 15 to 75 days post-inoculation. Tissue samples were collected and processed for light microscopy investigation. Gram-Chromotrope and Hematoxylin-Eosin staining techniques were performed, as well as GFAP and VIM immunohistochemical staining (avidin-biotin method). Infected mice presented multifocal granulomas in all organs, but the proportion of such granulomas was higher in group I. A lymphocytic, diffuse, non-suppurative menongoencephalomyelitis was observed and spores were seen in the microgranulomas. This astocytic response was more intense in group I than in group II and no VIM expression was seen in astrocytes from group III. The parasites were easily identified in Gram-Chromotrope stained sections. Objective: To evaluate an effectiveness of high-dose methylprednisolone therapy in patients with HTLV-I-associated myelopathy (HAM), we measured cerebrospinal fluid (CSF) neopterin concentrations as a marker which reflects degree of inflammation in central nervous system.Methods: Five HAM patients were treated with high-dose methylprednisolone (1000 mg/day i.v. CSF and peripheral blood were collected before and after the treatment. CSF neopterin levels were determined by high performance liquid chromatography. Peripheral blood mononuclear cells (PBMCs) were separated from peripheral blood by a density gradient method. Amount of HTLV-I proviral DNA were quantified by a real-time PCR. Clinical symptoms were evaluated using Osame Motor Disability Score and Urinary Disturbance Score.Results: CSF neopterin concentrations of all the patients were high (the mean value was 118 pmol/ml) exceeded a normal value (<20 pmol/ ml). The CSF neopterin levels were reduced after the high-dose methylprednisolone therapy (the mean value was 47 pmol/ml). The treatment also reduced the mean of HTLV-I proviral loads from 1084 to 769 copies per 10 4 PBMCs. Four out of five patients showed clinically improvement after the treatment.Conclusion: A high-dose methylprednisolone therapy is useful for patients with HAM. A CSF neopterin level is a valuable marker to evaluate the efficacy of treatments.Department of Microbiology and Immunology, and the Center for Molecular Virology and Neuroimmunology, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA, USA HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/ TSP) is characterized by the generation of an intense cytotoxic T cell (CTL) response with large numbers of CD8 + CTLs directed against oncoprotein Tax. Previous studies suggest that Tax may be available for immune recognition by dendritic cells (DCs), the most efficient antigen presenting cell population. In this study, we have shown that purified Tax protein efficiently bound and localized to the cell membrane of monocyte-derived dendritic cells (MDDCs) and was internalized within a few hours. After uptake, Tax induced expression of DC activation markers MHC class I and II, and costimulatory molecules (CD40, CD80, CD86, and CD54) as well as the DC maturation marker, CD83. Tax has also promoted the production of major immune-directing cytokines IL-12, TNF-α, and proinflammatory chemokines MIP-1α, MIP-1β, and RANTES, which drive a Th1-type immune response.These observations were also confirmed at gene level by microarray analyses. The inhibitors of NF-κB have abrogated Tax-induced secretion of cytokines/chemokines indicating a role for NF-κB signaling in the Tax-mediated immune response. Finally, Tax enhanced the allogenic and antigen-specific T cell proliferation capability of MDDCs. These results indicate that extracellular Tax may selectively target MDDCs, be taken up by these cells most likely by specialized pathways, and promote their maturation and antigen-presenting functions, driving a Th1-type immune response. PP21-21 Viral load determines the extent and quality of B cell responses in HIV infection of the central nervous system Sabine Cepok 1 , Gloria von Geldern 1 , Thorsten Nolting 1 , Verena Grummel 1 , Rajneesh Srivastava, Hans-Peter Hartung 1 , Ortwin Adams 2 , Gabriele Arendt 1 , Bernhard Hemmer 1 1 Department of Neurology, Heinrich Heine-University, Moorenstr. 5, 40225 Duesseldorf, Germany; 2 Department of Virology, Heinrich Heine-University, Moorenstr. 5, 40225 Duesseldorf, GermanyCentral nervous system (CNS) involvement is common in human immunodeficiency virus (HIV) infection. As the CNS constitutes an immunologically protected compartment, neurological progression is possibly independent of systemic viral burden. In HIV CNS infection an intrathecal Ig synthesis and pleocytosis are frequently observed. We analysed the humoral immune response in CSF and blood of HIV patients. 34 patients with HIV infection in different stages of disease were included in the study. We analyzed the B cell repertoire by four-colour flow cytometry and determined the viral load in CSF and blood of these patients. B cell subsets in HIV were compared to patients with other inflammatory or non-inflammatory neurological diseases. The number of B cells and plasmablasts was significantly elevated in CSF of HIV patients compared to both control groups. CSF B cell and plasmablast numbers were higher in untreated clinically asymptomatic HIV infected patients than symptomatic or AIDS patients. Determination of virus loads in both compartments revealed that CSF pleocytosis correlated with CSF virus titers but not with serum virus titers. In CSF and blood a strong correlation between the percentage of plasmablasts and HI virus load was observed. The extent of intrathecal production of IgG also correlated with CSF viral load and the percentage of plasmablasts. Our findings demonstrate a profound early B cell response to HIV in the CNS. Given the strong correlation between HI viral load and the occurrence of plasmablasts, these cells seem to be central in the humoral immune response to HIV in the CNS. PP21-22 HIV-1-infected and/or immune activated macrophages regulate astrocyte SDF-1 production through IL-1β H. Peng 1,2,3 , N. Whitney 1,2,3 , N. Erdmann 1,2,3 , A. Ghorpade 2,3,4 and J. Zheng 1,2,3,4 1 The Laboratory of Neurotoxicology at the 2 Center for Neurovirology and Neurodegenerative Disorders, 3 Department of Pharmacology/Experimental Neuroscience, and 4 Department of Pathology/Microbiology, University of Nebraska Medical Center, Omaha, NE 68198-5880, USA Stromal cell-derived factor 1 alpha (SDF-1α) and its receptor CXCR4 play important roles in the pathogenesis of HIV-1-associated dementia (HAD). However, the underlying mechanisms regulating SDF-1 production are unknown. In this report, we investigate the role of macrophage, an important cell type in HAD pathogenesis, in mediating SDF-1 production from astrocytes. Our data demonstrates that astrocytes are the major cell population expressing SDF-1 in the brain. Immune activated and/or HIV-1infected monocyte-derived-macrophage (MDM) conditioned media (MCM) induced a substantial increase in SDF-1 production by human astrocytes. Elevated SDF-1 production by astrocytes corresponds to a concomitant IL-1β upregulation in infected/activated macrophages. Moreover, MCMinduced astrocyte SDF-1 production is abrogated by IL-1β receptor antagonist (IL-1Ra) and IL-1β siRNA treatment of human MDM. These observations provide evidence that IL-1β from HIV-1-infected andactivated macrophage plays a critical role in SDF-1 production from astrocytes, and may contribute to SDF-1 mediated CNS regulation during HAD. Trafficking of monocytes through the blood brain barrier (BBB) is one pathway through which human immunodeficiency virus type 1 (HIV-1) can disseminate into the central nervous system (CNS). Interestingly, progenitor cells within the bone marrow are refractile to HIV-1 infection, but become susceptible as they differentiate. The TF-1 bone marrow progenitor cell line was selected as a model to study HIV-1 infection during the differentiation process of hematopoietic progenitor cells. The IL-1beta proinflammatory cytokine has been shown to have an effect on bone marrow progenitor cell proliferation. IL-1beta and a variety other of cytokines known to have a role in hematopoiesis (GM-CSF, M-CSF, IL-4) or inflammation (IL-6, TNF-alpha) were studied for their ability to induce differentiation. The effect of IL-1beta was considerably more dramatic than any other cytokine used resulting in CXCR4 and CCR5 upregulation with preservation of CD4 expression, potentially providing a window of opportunity for HIV-1 infection. CD34 and CD38 were downregulated while CD69 was modestly upregulated, indicative of differentiation and activation respectively. Moreover, transient and stable transfection analysis demonstrated that HIV-1 LTR activity was significantly increased following treatment of TF-1 cells with IL-1beta and TNF-alpha. These results indicate that progenitor cell differentiation potentially increases during the course of disease were evaluated, and their possible role as a predictive factor for neurological complications was statistically analyzed. Results: Seventy-six patients were male (67%) and sexual contact was the most common mode of transmission at 91.1%, with heterosexuality the dominating sexual preference (90.8%). Non-compliance to ART was associated with a higher incidence of neurological complications (p = 0.001, Chi-square test). Among all the variables investigated, only serum sodium levels at the time of diagnosis of HIV infection were found to be correlated with the development of neurological complications (p = 0.017, Mann-Whitney U test). Statistically significant declines in CD4 + and CD8 + T-cell counts were observed in association with neurological complications. Conclusion: Uninhibited HIV replication causes hyponatremia as a result of dysfunction of the renin-angiotensin system. The effect of cocaine on the expression of the chemokine receptor CXCR4 and the mechanisms by which cocaine regulates this receptor have yet to be determined in normal human astrocytes (NHA). Recent evidence indicates that cocaine activates mitogen-activated protein kinases (MAP kinases) to induce cellular changes. We hypothesize that cocaine alters signal transduction pathways in astrocytes leading to neuronal dysfunction in the infected subject. Treatment of NHA with cocaine increased the expression of CXCR4 and extracellular signal related kinase (ERK) as determined by quantitative PCR and western blot analyses. Furthermore, cocaineinduced phosphorylation of ERK occurred in a dose and time dependent manner (1 pM-1 μM, 15 min) with a maximum response occurring at 1 μM. No change in total ERK levels occurred. Exposure of NHA to PD 98059, an inhibitor of ERK, prior to cocaine treatment, prevented the upregulation of CXCR4 induced by cocaine alone. These results suggest that cocaine-induced modulation of neuroimmune pathogenesis of HIV-1 infection may be mediated via dysregulation of MAPK and may help to develop novel therapeutic strategies to prevent cocaine induced neuronal dysfunctions. PP21-29 Antiretroviral toxic neuropathy in lentivirus infection: Impaired brain-derived neurotrophic factor production and mitochondrial injury Background: Antiretroviral toxic neuropathy (ATN) is a common form of peripheral neuropathy in patients with HIV-1 infection although its pathogenesis is unclear. Herein, we developed a model of ATN induced by didanosine (ddI) following infection by the closely related lentivirus, feline immunodeficiency virus (FIV). Methods: After treatment with ddI of FIV-infected animals and dorsal root ganglia (DRG) cultures, we investigated neuronal and nerve morphology, neurobehavioral testing, viral load, mitochondrial and neurotrophic factor gene expression. Results: Both FIV and ddI caused titer and concentration-dependent DRG neuronal injury in vitro, respectively, with evidence of an additive neurotoxic effect depending on the neuronal parameter. In vivo FIV infection showed delays in latency to a noxious stimulus, which were exacerbated by ddI treatment. FIV-infected DRG neurons were highly vulnerable to ddI toxicity. The epidermal density of sural nerve endings were reduced after FIV infection, especially with ddI treatment. ddI did not diminish virus replication in DRGs of FIV-infected animals although it suppressed viral replication in sera from the same animals. ddI decreased mitochondrial cytochrome C oxidase unit II expression in DRGs of FIVinfected animals, and also in feline lymphocytes. Brain-derived neurotrophic factor (BDNF) expression was down-regulated by ddI in Schwann cells of DRGs following FIV infection. Conclusion: ddI treatment results in ATN, which was associated with mitochondrial injury and reduced BDNF production by Schwann cells. Hence, BDNF reduction may serve as a potential target for ATN therapy in the future. PP21-30 The involvement of gut-associated lymphoid tissues in TSE agent neuroinvasion from the intestine Bridget R. Glaysher and Neil A. Mabbott Institute for Animal Health, Ogston Building, West Mains Road, Edinburgh EH9 3JF, United Kingdom Neil.mabbott@bbsrc.ac.uk Transmissible spongiform encephalopathies (TSE) (or 'prion diseases') are neurodegenerative diseases that affect both humans and animals. Natural TSE infections are usually acquired by peripheral exposure, eg: ingestion or via skin lesions. Following exposure, infectivity usually accumulates in lymphoid tissues before spreading to the brain. We have shown that follicular dendritic cells (FDCs) resident in lymphoid follicles are important sites of TSE agent accumulation in lymphoid tissues. Indeed, TSE pathogenesis is critically dependent on FDCs as in their absence, TSE agent accumulation in lymphoid tissues is blocked and disease susceptibility is significantly reduced. Once TSE agents have accumulated on FDCs, infection spreads along peripheral nerves to the central nervous system, a process termed 'neuroinvasion'. However, it is not known from which lymphoid tissue/s TSE agent neuroinvasion actually occurs from. For example, following oral inoculation, PrP Sc accumulates first on FDCs in Peyer's patches and gut-associated lymphoid tissues and subsequently spreads to nondraining lymph nodes and the spleen. Whether efficient neuroinvasion occurs directly following accumulation upon the FDCs that are first encountered within the draining lymphoid tissue (eg: Peyer's patches following oral inoculation) or requires dissemination from FDCs networks within multiple lymphoid tissues is not known. Using mice deficient in various combinations of gut-associated lymphoid tissues we investigate the contribution of these tissues to TSE agent neuroinvasion from the intestine following oral exposure. The identification of the cells, tissues and mechanisms involved in TSE agent neuroinvasion may identify an important process to which therapeutic intervention can be directed.with therapy resistant localisation-related epilepsy. The patients were categorised TLE + HS (N = 16), TLE − HS (N = 16) and extratemporal epilepsy (N = 16) based on ictal eletro-clinical characteristics and high resolution MRI. Patients with suspected CD or gluten sensitivity underwent duodenal biopsies. Results: Seven patients were gluten sensitive, all of these patients had TLE + HS whereas none of the patients without HS were gluten sensitive (p < 0.0002). In duodenal biopsies three of the patients had histological evidence of CD and four had inflammatory changes consistent with early developing CD without villous atrophy. Four of the patients with gluten sensitivity had evidence of dual pathology (HS+ another brain lesion) whereas none of the rest of patients did (p < 0.0002). Conclusion: The present study demonstrates a previously unrecognized association between gluten sensitivity and TLE with hippocampal sclerosis. The association was very robust in this well characterised group of patients; thus gluten sensitivity should be added to the list of potential mechanism leading to intractable epilepsy and HS. PP22-05 Type I diabetes mellitus and central nervous system demyelinization: A possible association Asli Kurne, Eser Sevgi Basak, Rana Karabudak Hacettepe University School of Medicine, Department of Neurology, Ankara, TurkeyObjective: Autoimmune diseases are a heterogeneous group of disorders leading to the disruption of the body's own tissues by the immune system. Recent epidemiological studies have shown an increased risk of acquiring a new autoimmune disease for subjects having one autoimmune disease. Multiphasic demyelinating autoimmune syndromes and MS can occur concurrently with other autoimmune diseases including autoimmune thyroid disease, autoimmune gastritis, Addison's disease, rheumatoid arthritis, pemphigus vulgaris and scleroderma. In different populations an increased prevalence of multiple sclerosis in adults with type 1 diabetes and their firstdegree relative has been reported before. Method-results: We present three patients with type 1 diabetes mellitus who developed central nervous system demyelinating disease in their follow-up. PP22-06 Myelopathy in Sjögren syndrome: Report of 7 cases activation, but very little evidence of Aβ phagocytosis. These observations are in contrast to previous evidence that microglial activation in neurodegenerative disorders is only harmful, and suggest that microglial activation represents a two-edged sword with both harmful and potentially beneficial effects. PP23-06 Effect of T cell sensitization on injured motoneurons following facial nerve axotomy in the adult mouse Recent studies suggest that T cells can attenuate the levels of motoneuron degeneration following facial nerve axotomy in adult mice. An important feature of T cells is their ability to become sensitized to specific antigens such that re-exposure to an antigen produces a more robust T cell response. We hypothesized that T cells with prior exposure to motoneuron injury would show increased responsiveness to subsequent motoneuron injury and that these sensitized T cells would enhance neuronal survival. Naïve mice received a sham surgery while sensitized mice received an axotomy of the facial nerve. Both groups received an experimental axotomy of the contralateral nerve 9.5 weeks later. The neuroimmune response was quantified in the experimentally axotomized facial motor nucleus (FMN) for numbers of CD3 + T cells, microglia expressing major histocompatibility complex II, and CD11b + microglial phagocytic clusters. The effect of T cell sensitization on motoneuron viability following injury was determined by counting the number of surviving motoneurons and by measuring the cross-sectional area of motoneurons in representative sections throughout the FMN. The number of T cells was greater in the injured FMN of sensitized mice following the experimental axotomy compared to naïve controls. The enhanced T cell response was not accompanied by improved motoneuron survival. Our data suggest that T cell sensitization occurs in response to neuronal injury and that the increased responsiveness of these cells is not sufficient to promote motoneuron survival. Further research is required to determine whether T cells can alter the functional status of neurons following injury. PP23-08 Nitric oxide exposure diverts neural stem cell fate from neurogenesis towards astrogliogenesis Aim: To study the effect of nitric oxide (NO) on differentiation pattern of adult neural stem cells (NSC). Introduction: In the central nervous system (CNS) NO regulates the proliferation of neural stem/progenitor cells both during development and adulthood. The effects of NO on differentiation were assessed by immuno-labeling the cells for neuronal (Tuj), oligodendrocyte-(O4) and astrocyte-(GFAP) lineage markers and with western blotting. Results: After exposure to NO, neurogenesis was downregulated and this corresponded to decreased expression of the pro-neural gene neurogenin-2, and β-III-tubulin. The decreased ability to generate neurons was also found to be transmitted to the progeny of the cells. NO exposure was instead beneficial for astroglial differentiation, which was confirmed by increased activation of the JAK/STAT-1 signal transduction pathway. Conclusion: These results have clinical implications for diseases like Multiple Sclerosis and trauma, where neuroregeneration is impaired by glial scar formation. PP23-09 Age and injury-related molecular profiling of the subgranular zone (SGZ) in CD1 mice: Contribution to hippocampal injuryinduced neurogenesis The hippocampal dentate gyrus contains neural progenitors that continue to proliferate and differentiate into both glia and neurons. Active maintenance of this cell population declines with aging, which may contribute to decreased repair and plasticity. The environment of the proliferative zones modulates neurogenesis with the critical factors yet to be identified. From CD-1 mice (22 days or 1 year old), the SGZ of the hippocampus was isolated by laser capture microdissection, RNA amplified and hybridized to Affymetrix Whole Mouse Genome GeneChips. The molecular profile differed as a function of age with 398 genes/ESTs showing differentially altered expression. When the SGZ was examined following injury induced by the hippocampal toxicant, trimethyltin, levels for 1083 genes/ESTs were altered. Differential responses with injury occurred as a function of age. Pathway mapping of the molecular profile in the neurogenic environment included the interleukin-1α pathway (IL1A, MYD88, TRADD, and TNFRSF1A) in the immature brain and the IL-6 pathway (IL6, IL6, Grb2, RAF1, and MAPK1) in the 1-yearold brain. This pathway difference was seen in both naïve and injured brain. The level of neurogenesis induced by TMT, as identified by BrdU + /NeuN + immunostaining, suggests a greater response in the 22-day-old mouse as compared to the response at 1-year. Characterization of the age related differences in the neurogenic environment will identify novel signaling factors to promote repair in the adult brain.Supported by Intramural Research Program, NIEHS/NIH PP23-10 Neural differentiation potential of mesenchymal stem cells derived from bone marrow, fat, spleen and thymus Mesenchymal stem cells (MSC) are multipotent elements with the capacity to differentiate in vitro into several cell lineages. Conflicting results have been reported regarding their neural differentiation potential.To evaluate the neural differentiation potential of human MSC derived from BM, fat, spleen and thymus in vitro and in co-cultures with glial cells.MSC were derived from healthy human donors and Schwann cells were cultured from human benign schwannomas according to standard protocols [1, 2] .MSC displayed a neural morphology and phenotype after 30 h with a specific differentiation protocol [3] . Differentiated cells expressed neuronal and glial markers, such as NeuN, MAP2, PSA-NCAM, PMP22, S100, GFAP and GalC. This differentiation was transient and MSC returned toWe have previously demonstrated that human mesenchymal cells (MSC) inhibit T and B cells proliferation arresting them in the G0/1 phase of the cell cycle. Such effect is not associated with the induction of apoptosis as the percentage of apoptotic lymphocytes upon antigen receptor activation in the presence of MSC was negligible. Thus, we sought to study the effect of MSC on T cells and B cells undergoing apoptosis by different stimuli. MSC rescued T and B cells from death when cultured in serum free condition. Similar effect was observed on human isolated thymocytes that were rescued from spontaneous apoptosis. MSC also decreased the percentage of annexin V positive T cells upon the induction of activation induced cell death (AICD). Upon AICD trigger in the presence of MSC, we observed a decreased expression of Fas and FasL, granzyme B and total caspases by T cells. MSC also decreased anti-Fas induced apoptosis of Jurkat T-cells. To study the effect of MSC on B cells apoptosis, we isolated normal germinal center (GC) B cells, a subset undergoing spontaneous apoptosis in secondary lymphoid organs, which can be reverted in vitro by CD40 ligation. MSC rescued GC B cells from spontaneous apoptosis with an efficiency comparable to that of CD40L. These findings are relevant for the use of MSC for multiple sclerosis and other autoimmune diseases as they suggest that MSC have an immunosuppressive effect on lymphocyte proliferation but support their survival in a quiescent state and protect them from apoptosis. PP23-13 Temporal expression of integrins and chemokine receptors in adult neuronal stem cells and partial analysis of intra-cellular signaling pathway R. Zilkha-Falb, N. Kaushansky and A. Ben-Nun Department of Immunology, The Weizmann Institute of Science, Rehovot, 76100, IsraelThe therapeutic potential of adult neuronal stem cells (adultNSC) has been suggested in several models of central nervous system (CNS) insult. We therefore investigated the expression (RT-PCR) of integrins and chemokine receptors before and after in-vitro differentiation of adultNSC, their potential involvement in adhesion and migration of adultNSC, and their homing to insulted areas in the brain. Our analysis of several members of integrins and of CCR and CXCR chemokine receptors show that adultNSC express a relatively high basal level of VLA-1 and CXCR4, which are significantly upregulated upon induction of differentiation. CXCR5 is constitutively expressed, albeit in very low levels, and is not upregulated upon adultNSC differentiation. CXCR1 and CXCR3 are not expressed in the undifferentiated adultNSC, but are significantly induced upon differentiation. Analysis of intra-cellular signaling pathways activated by the ligands of the integrins, in particular laminin, suggests the involvement of the ERK pathway and of the upstream signaling mediators Fyn and Grb2. In contrast, activation by the CXCL12 and CXCL13 ligands of CXCR4 and CXCR5, respectively, reduced ERK phosphorylation but increased p38 phosphorylation.These results suggest the involvement of the ERK signaling pathway in adultNSC activation by ligands of the integrins that we tested, and of the p38 signaling pathway in activation by CXCL12 and CXCL13 chemoattractants. PP23-14 Identification of signaling pathways involved in innate immune responses in human embryonic stem cells A. Campbell and H. Keirstead University of California, Irvine, California, USAIn the developing spinal cord, motoneurons and oligodendrocytes are produced from the ventral ventricular zone, called the pMN domain. Olig2 is a basic helix-loop-helix (bHLH) transcription factor which is essential for the generation of motoneurons and oligodendrocytes. Besides, previous lineage analysis experiments using Olig2-GFP and Olig1-Cre mice suggested that Olig2-expressing progenitors in the pMN domain generate motoneurons and oligodendrocytes, but not astrocytes. So, Olig2-expressing progenitors are expected as possible candidates for the transplantation therapy in demyelinating diseases, such as multiple sclerosis, or spinal cord injury. However, previous lineage analysis systems include some problems, it is not clear whether another cell-type is produced from the pMN domain. For clinical application of Olig2-expressing progenitors, it is very important to reveal whether motoneurons and oligodendrocytes are the only cell types of cells produced from the pMN domain. To elucidate the accurate lineage of Olig2-expressing progenitors, we performed lineagetracing experiments using a tamoxifen-inducible Cre-recombinase inserted into the Olig2 locus. In this study, we demonstrated that motoneurons and oligodendrocytes are derived from the Olig2-expressing progenitors in the pMN domain, and also found that a subset of astrocytes and ependymal cells are also produced from the pMN domain. Our system will aid elucidation of the molecular mechanisms of cell specification in this lineage. PP23-19 Pre-existing immunity inhibits expression of transgene in muscle cells of immune competent animals Host immune response is one of the critical factors for the success of gene therapy. In addition to immunity against the vial vectors, the mutant hosts also develop immune responses to the transgenic products that are nonimmunogenic in normal animals. Methods: Fifteen adult female Balb/c mice were divided into three groups and immunized intradermally. The animals were immunized four times. Seven days after the last immunization, they received intramuscular injection of pCBLacZ. Result: ELISA for the titers of anti-galactosidase IgG1 and IgG2a showed that the animals treated with protein developed a Th2 predominant immunity and the mice receiving DNA had a preferential Th1 response. Mean numbers of muscle cells expressing beta-galactosidase were much less in the animals immunized with protein or DNA as compared with the little mates. Those were 0, 0.8 and 23.6. Discussion: Gene delivery to muscle is a potential therapy for protein deficiencies such as hemophilia. Patients may have ever received protein therapy years before the application of gene therapy, or they may need repeated gene therapy to keep long-term expression of the transgene. In this study, we demonstrate that pre-existing immunity with either Th1 or Th2 predominance inhibits transgene expression in muscle cells. PP23-20 Intrasplenic electro-transfer of IL-4 encoding plasmid DNA efficiently inhibits rat experimental allergic encephalomyelitis Seong-Hyun Ho 1,3 , Hwang-Jae Lee 1 , Dong-Sik Kim 1 , Jae-Gyun Jeong 1 , Sujeong Kim 1 , Seung Shin Yu 2 , Sunyoung Kim 1,3 , Jong-Mook Kim 1